scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

scholarly journals Direct PCR amplification from saliva sample using non-direct multiplex STR kits for forensic DNA typing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pankaj Shrivastava ◽  
Toshi Jain ◽  
R. K. Kumawat
Keyword(s):  
Dna Typing ◽  
Pcr Amplification ◽  
Turnaround Time ◽  
Forensic Dna ◽  
Direct Pcr ◽  
Forensic Dna Typing ◽  
Dna Profile ◽  
Pre Treatment ◽  
Dna Profiles ◽  
Direct Amplification

AbstractDue to its proficiency to provide the most discriminating results for forensic applications, medical research and anthropological studies, multiplex PCR based STR analysis has been established as the most efficient technique in the forensic DNA analysis. Several multiplex amplification kits based on 4, 5 and 6 dyes chemistry are commercially available and used in forensic DNA typing across the globe. These multiplex PCR systems are routinely used for amplification of multiple STR loci (Autosomal, Y and/or X STR’s) in the DNA extracted from various biological samples. In the routine forensic DNA testing, DNA profile obtained is compared with the DNA profile of the reference sample, which takes a certain turnaround time and employs costly lab resources. Successive development in forensic DNA typing have resulted in advent of improved multiplex kits which have reduced the effective analysis time, cost and minimized the number of steps required in comparison to conventional forensic DNA typing. Specialized direct amplification compatible multiplex kits are also available nowadays. These kits are relatively costlier but still require few pre-processing steps, which does not make them worth the hefty cost. Herein, this study, we have used non-direct multiplex STR kits to assess their efficacy for direct amplification. In the present study, 103 saliva samples were directly amplified without any pre-treatment of the samples using thirteen non-direct multiplex kits (4 dyes, 5 dyes and 6 dyes chemistry based) for forensic DNA typing. Here, we report a validated direct PCR amplification protocol from the reference saliva samples by omitting DNA extraction and quantification steps, which resulted in 80% reduction of the turnaround time. The developed protocol is cost effective, time efficient and it does not compromise with the quality of DNA profiles. To the best of our knowledge, this is the first report for direct amplification of DNA with the most commonly used non-direct multiplex STR kits without any pre-treatment of the sample. Complete DNA profiles matching all the essential quality parameters were obtained successfully from all the tested samples.

scholarly journals Optimisation of rapid STR analysis using a standard DNA forensic pipeline

2019 ◽  
Author(s):  
Katharine Gammon ◽  
Carl Mayers
Keyword(s):  
Capillary Electrophoresis ◽  
Pcr Amplification ◽  
Buccal Cell ◽  
Forensic Dna ◽  
Direct Pcr ◽  
Str Analysis ◽  
Dna Profile ◽  
Fta Cards ◽  
Dna Profiles ◽  
Reference Samples

Previous studies in published literature have reported on various alterations to STR mastermixes, protocols and instrumentation in order to reduce the time taken to generate forensic DNA profiles from reference and casework type samples. In this study, we demonstrate how altering default PCR amplification and capillary electrophoresis protocols in our existing DNA profiling pipeline can reduce the overall time taken to generate a DNA profile from buccal cell reference samples. GlobalFiler Express STR mastermix was used with direct PCR from FTA cards, run on altered PCR protocols and CE settings, and results compared to the standard evaluated settings used in our laboratories. This study demonstrated that full DNA profiles could be recovered in less than 80 minutes in comparison to our standard time of 97 – 102 minutes whilst utilising existing reagent kits and instrumentation, with only minor modifications to protocols.

Detection ofTrypanosoma congolenseandTrypanosoma bruceisubspecies by DNA amplification using the polymerase chain reaction

Parasitology ◽  
1989 ◽  
Vol 99 (1) ◽  
pp. 57-66 ◽  
Author(s):  
D. R. Moser ◽  
G. A. Cook ◽  
Diane E. Ochs ◽  
Cheryl P. Bailey ◽  
Melissa R. McKane ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Large Scale ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Detection Methods ◽  
Chain Reaction ◽  
African Trypanosomes ◽  
Polymerase Chain

SUMMARYThe nuclear DNA ofTrypanosoma congolensecontains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA ofTrypanosoma brucei brucei(Sloofet al.1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite ofT. congolenseorT. bruceispp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor inLeishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected withT. congolenseand/orT. bruceispp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

scholarly journals Comparison of Effects of Acidic Electrolyzed Water and NaOCl on Tilletia indica Teliospore Germination

Plant Disease ◽  
1999 ◽  
Vol 83 (7) ◽  
pp. 627-632 ◽  
Author(s):  
M. R. Bonde ◽  
S. E. Nester ◽  
A. Khayat ◽  
J. L. Smilanick ◽  
R. D. Frederick ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Alternative Methods ◽  
Karnal Bunt ◽  
Tilletia Indica ◽  
Chain Reaction ◽  
Direct Pcr ◽  
Electrolyzed Water ◽  
Teliospore Germination ◽  
Polymerase Chain ◽  
Axenic Cultures

Definitive identification of free teliospores of Tilletia indica, causal agent of Karnal bunt of wheat, requires polymerase chain reaction (PCR)-based diagnostic tests. Since direct PCR amplification from teliospores has not been reliable, teliospores first must be germinated in order to obtain adequate DNA. We have routinely surface-sterilized teliospores for 2 min with 0.4% (vol/vol) sodium hypochlorite (NaOCl) to stimulate germination and produce axenic cultures. However, we observed that some spores were killed even with a 2-min NaOCl treatment, the shortest feasible duration. Decreasing the NaOCl concentration in our study from 0.4% to 0.3 and 0.2%, respectively, increased teliospore germination, but treatment times longer than 2 min still progressively reduced the germination percentages. In testing alternative methods, we found “acidic electrolyzed water” (AEW), generated by electrolysis of a weak solution of sodium chloride, also surface-sterilized and increased the rate of T. indica teliospore germination. In a representative experiment comparing the two methods, NaOCl (0.4%) for 2 min and AEW for 30 min increased germination from 19% (control) to 41 and 54%, respectively, by 7 days after treatment. Because teliospores can be treated with AEW for up to 2 h with little, if any, loss of viability, compared with 1 to 2 min for NaOCl, treatment with AEW has certain advantages over NaOCl for surface sterilizing and increasing germination of teliospores of suspect T. indica.

scholarly journals Diagnosis of blackleg from cattle tissue impregnated in common filter paper

2021 ◽  
Vol 51 (8) ◽  
Author(s):  
Julia Pires Espíndola ◽  
Luana D’Avila Farias ◽  
Cláudia Balzan ◽  
Valessa Lunkes Ely ◽  
Agueda Palmira Castagna de Vargas
Keyword(s):  
Filter Paper ◽  
Tissue Type ◽  
Limiting Factor ◽  
Direct Pcr ◽  
Tissue Storage ◽  
Tissue Samples ◽  
Polymerase Chain ◽  
Clostridium Chauvoei ◽  
Economical Alternative

ABSTRACT: Blackleg, an acute myonecrosis caused by Clostridium chauvoei, is usually underdiagnosed since the rapid transport of adequate samples for laboratory testing is difficult. This study tested a direct polymerase chain reaction (PCR) technique using common filter paper impregnated with cattle tissue samples obtained from animals suspected with blackleg. Twenty-five samples, belonging to eleven animals from Rio Grande do Sul State, Brazil, were analyzed. The direct PCR technique identified eight positive animals corroborating with results from microbiological culture. Skeletal muscle was the most common tissue type used in this study and when the animal was positive the pathogen was always detected in this tissue. Storage time of the impregnated filter paper at room temperature did not prove to be a limiting factor for the quality of the results indicating that this procedure can be carried out in the field and samples be sent in regular mail. Our results suggested that direct PCR of common filter paper impregnated with cattle tissue is a practical and economical alternative for the diagnosis of blackleg.

scholarly journals A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

2021 ◽  
Vol 9 (12) ◽  
pp. 2505
Author(s):  
Hiroki Hayashi ◽  
Tsutomu Kishi
Keyword(s):  
Dna Sequences ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Epitope Tagging ◽  
One Step ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The One ◽  
Step Polymerase Chain Reaction

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

scholarly journals Clonal Stability of RAPD Markers in Three Rhododendron Species

1995 ◽  
Vol 13 (1) ◽  
pp. 43-46 ◽  
Author(s):  
M. Javed Iqbal ◽  
D. W. Paden ◽  
A. Lane Rayburn
Keyword(s):  
Dna Sequences ◽  
Rapd Markers ◽  
Cultivar Identification ◽  
Rapd Analysis ◽  
Randomly Amplified Polymorphic Dna ◽  
Plant Identification ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
The Individual ◽  
Species Specific

Abstract Amplification profiles produced by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA sequences (RAPD) have the potential for species and cultivar identification. Since most rhododendron plants are vegetatively propagated, it is imperative that RAPD profiles be stable during this propagation. Three species of rhododendron, Rhododendron arborescens, R. atlanticum and R. yedoense var. poukhanense were used to produce species specific amplification profiles. Stability of amplification profiles among individually cloned plants of each species were studied. Ten plants of R. atlanticum, 9 of R. arborescens, and 10 of R. yedoense var. poukhanense were studied with 10 random primers. No polymorphism was observed among individual plants of R. atlanticum and R. arborescens with all the primers. The amplification product of one plant of R. yedoense var. poukhanense showed a difference of one band with one primer. The rest of the profiles with 9 primers were identical in all plants of this species. In order to ascertain that RAPD markers can indeed reveal real genetic differences among plants, F2 plants of two hybrids were analyzed. In contrast to the clonally propagated plants, extensive polymorphisms were observed among the individual F2 plants. Thus, RAPD analysis can be used to detect genetic variability. This stability of RAPD profiles in clonally propagated rhododendron indicates the usefulness of these markers in plant identification.

Differentiation of isolates in the genus Steinernema (Nematoda: Steinernematidae).by random amplified polymorphic DNA fragments and morphological characters

Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J. Liu ◽  
R. E. Berry
Keyword(s):  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Amplification ◽  
Nematode Species ◽  
Morphological Characters ◽  
Dna Fragments ◽  
Undescribed Species ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
Polymerase Chain

SUMMARYWe combined polymerase chain reaction (PCR) amplification of DNA sequences and important morphological characters as a technique to differentiate nematode isolates in the genus Steinernema. Five decamer oligonucleotide primers were used to generate random amplified polymorphic DNA (RAPD) fragments from 11 nematode isolates. The primers generated 8–12 fragments, ranging from 220 to 1300 bp in size. Reproducible amplified DNA fragments of 11 isolates showed obviously inter- or intra-specific polymorphisms, enabling us to differentiate easily the nematode species and isolates. Combining RAPD–PCR fragments with the examination of morphological characters of infective juveniles and 1st-generation males, we identified isolate OH1S, collected from Newport, Oregon, as S.feltiae; isolate OS21, collected from Grants Pass, Oregon, belonged to a previously undescribed species. Our study may provide a rapid and reliable method for the identification of Steinernema nematodes.

scholarly journals UMIc: A Preprocessing Method for UMI Deduplication and Reads Correction

2021 ◽  
Vol 12 ◽  
Author(s):  
Maria Tsagiopoulou ◽  
Maria Christina Maniou ◽  
Nikolaos Pechlivanis ◽  
Anastasis Togkousidis ◽  
Michaela Kotrová ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Library Preparation ◽  
Chain Reaction ◽  
Consensus Sequences ◽  
Alignment Free ◽  
Unique Identity ◽  
Polymerase Chain ◽  
Recent Refinement

A recent refinement in high-throughput sequencing involves the incorporation of unique molecular identifiers (UMIs), which are random oligonucleotide barcodes, on the library preparation steps. A UMI adds a unique identity to different DNA/RNA input molecules through polymerase chain reaction (PCR) amplification, thus reducing bias of this step. Here, we propose an alignment free framework serving as a preprocessing step of fastq files, called UMIc, for deduplication and correction of reads building consensus sequences from each UMI. Our approach takes into account the frequency and the Phred quality of nucleotides and the distances between the UMIs and the actual sequences. We have tested the tool using different scenarios of UMI-tagged library data, having in mind the aspect of a wide application. UMIc is an open-source tool implemented in R and is freely available from https://github.com/BiodataAnalysisGroup/UMIc.

scholarly journals A Simple Gold Nanoprobe Assay for the Identification of Staphylococcus Aureus, Listeria Monocytogenes and Salmonella Enteritidis in Food Specimens

2017 ◽  
Vol 6 (4) ◽  
pp. 134 ◽  
Author(s):  
Dimitra Panagiotis Houhoula ◽  
Ekatherina Charvalos ◽  
Spyros Konteles ◽  
Stamatis Koussissis ◽  
Vladimiros Lougovois ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
Dna Sequences ◽  
Salmonella Enteritidis ◽  
Screening Method ◽  
Pcr Amplification ◽  
Reference Method ◽  
Direct Pcr ◽  
Salmonella Spp ◽  
Gold Nanoprobe

In the present study, we developed a gold nanoprobe assay, which relies on the colorimetric differentiation of specific DNA sequences, based on different aggregation profiles. We evaluated the assay on DNA extracted from pathogen cultures and from contaminated food specimens. The targets used were the femA gene for the identification of Staphylococcus aureus, the hly gene of the Listeria monocytogenes listeriolysin and the invA sequence for Salmonella spp. Comparison was performed with the reference assay, as described in the respective ISO guidelines for each pathogen, and a direct PCR amplification and detection. The minimum detection limit of the assay was defined at 123 fg/μL DNA, for all species, lower than PCR detection. Specificity was 100%. Sensitivity was 100%, as compared το the reference method, whereas the sensitivity of PCR was 93.3%. The evaluated assay could be used as a sensitive screening method, which does not require major infrastructure, for the detection and identification of pathogens in food specimens. In addition, it can accommodate detection of multiplespecies,thus allowing multiplexingand expedite turnaround time. 

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