scholarly journals Efficiency of Randomly Amplified Polymorphic DNA to Sequence Characterized Amplified Region Marker Conversion and their Comparative Polymerase Chain Reaction Sensitivity in Cucumber

1999 ◽  
Vol 124 (2) ◽  
pp. 128-135 ◽  
Author(s):  
Thomas Horejsi ◽  
Jodie M. Box ◽  
Jack E. Staub
Keyword(s):  
Rapd Markers ◽  
Scar Marker ◽  
Scar Markers ◽  
Sequence Characterized Amplified Region ◽  
Pcr Product ◽  
Rapd Pcr ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Template Dna ◽  
Pcr Conditions

The conversion of randomly amplified polymorphic DNA (RAPD) markers to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions were studied in cucumber (Cucumis sativus L.). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16 to 22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64%) of 75 RAPD markers were successfully converted to SCAR markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer pairs produced multiple polymorphic PCR products. The band intensity of SCAR markers were brighter (P = 0.05) than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced (P = 0.05) by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities than their RAPD counterparts and SCAR markers tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mm) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type of thermocycler used affected the performance of SCAR and RAPD markers. These data suggest that although SCAR markers may demonstrate enhanced performance over the RAPD markers from which they are derived, careful consideration must be given to both the costs and potential benefits of SCAR marker development in cucumber.

scholarly journals PCR-based molecular markers for identification of taxa from the Calypogeia fissa complex (Jungermanniopsida, Calypogeiaceae)

2012 ◽  
Vol 28 (1) ◽  
pp. 9-18 ◽  
Author(s):  
Katarzyna Buczkowska ◽  
Patrycja Gonera ◽  
Bartosz Hornik
Keyword(s):  
Molecular Markers ◽  
Dna Sequences ◽  
Scar Marker ◽  
Issr Markers ◽  
Genetic Differences ◽  
Scar Markers ◽  
Sequence Characterized Amplified Region ◽  
Pcr Product ◽  
Isozyme Loci ◽  
Diagnostic Traits

Abstract Within Calypogeia fissa, two subspecies connected with geographic distribution are formally recognized: C. fissa subsp.fissa in Europe and C. fissa subsp.neogea in North America. Isoenzyme studies have shown that the European subspecies is genetically differentiated and composed of three genetically distinct groups PS, PB and G. The PS group has the most distinctive morphological features, but no morphological diagnostic traits have been found for groups PB and G. The sequence characterized amplified region (SCAR) markers developed on the basis of ISSR markers, applied in the study, allowed the delimitation of all groups distinguished in Europe within the C. fissa complex (PS, PB and G). The markers also revealed genetic differences between the European and American subspecies. Five primer pairs (Cal01, Cal03-Cal06) of the six pairs studied are useful as the diagnostic tool for the identification of particular groups from the C.fissa complex. The examined SCAR markers showed that the PS group of C.fissa subsp.fissa was the most distinct; it differed from both groups PB and G as well as from C.fissa subsp.neogea. All plants determined on the basis of diagnostic isozyme loci as the PS group amplified a longer product (380 bp) of the Cal04 primer pair than the rest of studied groups and yielded no amplification products in Cal03, Cal05 and Cal06 primers. The primer pair Cal03 distinguished the plants of the PB group from the remaining groups, since only the PB group generated a PCR product of about 290 bp. The genetic differences between all four studied groups of the C.fissa complex were supported by DNA sequences of the SCAR marker Cal04.

scholarly journals Parallel DNA polymerase chain reaction: Synthesis of two different PCR products from a DNA template

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 320 ◽  
Author(s):  
Vikash Bhardwaj ◽  
Kulbhushan Sharma
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Chain Reaction ◽  
Single Stranded Dna ◽  
Pcr Product ◽  
Pcr Products ◽  
Parallel Direction ◽  
Polymerase Chain ◽  
Dna Template ◽  
Template Dna

Conventionally, in a polymerase chain reaction (PCR), oligonucleotide primers bind to the template DNA in an antiparallel complementary way and the template DNA is amplified as it is. Here we describe an approach in which the first primer binds in a parallel complementary orientation to the single-stranded DNA, leading to synthesis in a parallel direction. Further reactions happened in a conventional way leading to the synthesis of PCR product having polarity opposite to the template used. This is the first study showing that synthesis of DNA can happen also in a parallel direction. We report that from a single-stranded DNA template, two different but related PCR products can be synthesized.

scholarly journals Optimization of the RAPD Marker Techniques in Camellia

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 973D-973
Author(s):  
Lianghong Chen ◽  
Mack Nelson
Keyword(s):  
Annealing Temperature ◽  
Rapd Marker ◽  
Dna Amplification ◽  
Breeding Programs ◽  
Rapd Pcr ◽  
Polymerase Chain ◽  
Template Dna ◽  
Pcr Conditions ◽  
Reaction Component ◽  
Single Set

Randomly amplified polymorphic DNA (RAPD) technique is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. It has been widely used for genetic mapping, plant and animal breeding programs, and DNA fingerprinting. However, there is no single set of RAPD-PCR conditions that can be applied to all situations. In order to adjust reaction component concentrations within suggested ranges for efficient amplification during the use of RAPD in detection of genetic variation of genus Camellia, crucial factors, such as concentrations of MgCl2 and DNA, annealing temperature (37 to 44 °C), and the use of an AmpliTaq® DNA polymerase and Stoffel fragment were examined. Five camellia cultivars, `Winter's Beauty', `Pink Icicle', `Polar Ice', `Winter's Hope', and `Snow Flurry', were under investigation. Clear and reproducible amplification products were produced with 3.0 μM MgCl2 and 30 ng template DNA/25 μL reaction mixer at annealing temperature 37 °C and 40 °C, compared with MgCl2 at 1.5, 2.0, and 2.5 μM. When annealing temperature increased, the RAPD-PCR stringency was increased, as expected. Stoffel fragment was found to provide highly reproducible results.

scholarly journals A PCR-Based Method of Detection and Differentiation of K88+ Adhesive Escherichia Coli

1996 ◽  
Vol 8 (4) ◽  
pp. 460-463 ◽  
Author(s):  
Mark A. Franklin ◽  
David H. Francis ◽  
Diane Baker ◽  
Alan G. Mathew
Keyword(s):  
Escherichia Coli ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigenic Variant ◽  
Variable Regions ◽  
E Coli ◽  
Clinical Assay ◽  
Pcr Product ◽  
Structural Subunit ◽  
Pcr Products ◽  
Polymerase Chain

The objective of this study was to develop a polymerase chain reaction (PCR)-based method to detect and differentiate among Escherichia coli strains containing genes for the expression of 3 antigenic variants of the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers were designed that allowed detection of K88+ E. coli, regardless of antigenic variant, and the separate detection of the ab, ac, and ad variants. Primers AM005 and AM006 are 21 base pair (bp) oligomers that correspond to a region of the K88 operon that is common to all 3 antigenic variants. Primers MF007, MF008, and MF009 are 24-bp oligomers that matched variable regions specific to ab, ac, and ad, respectively. Using primers AM005 and AM006, a PCR product was obtained that corresponds to a 764-bp region within the large structural subunit of the K88 operon common to all 3 antigenic variants. Primer AM005 used with MF007, MF008, or MF009 produced PCR products approximately 500-bp in length from within the large structural subunit of the K88 operon of the 3 respective antigenic variants. Fragments were identified by rates of migration on a 1% agarose gel relative to each other as well as to BstEII-digested lambda fragments. This PCR-based method was comparable to the enzyme-linked immunosorbent assay and western blot test in the ability to differentiate between the antigenic variants. K88+ E. coli were differentiated from among laboratory strains and detected in ileal samples taken from cannulated pigs challenged with a known K88+ variant. K88+ E. coli were also detected from fecal swabs taken from newly weaned pigs, thus confirming that this PCR-based test could provide a convenient clinical assay for the detection of K88+ E. coli. Detection and differentiation of K88+ E. coli using general and specific primers was successful. PCR methods of detection should permit identification of K88+ antigenic variants regardless of the level of expression of the antigen.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

1993 ◽  
Vol 39 (9) ◽  
pp. 1927-1933 ◽  
Author(s):  
J B Findlay ◽  
S M Atwood ◽  
L Bergmeyer ◽  
J Chemelli ◽  
K Christy ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Automated System ◽  
Closed Vessel ◽  
Chain Reaction ◽  
Panel Testing ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

Genetic diversity among microsporidian isolates from the silkworm, Bombyx mori, as revealed by randomly amplified polymorphic DNA (RAPD) markers

2011 ◽  
Vol 56 (4) ◽  
Author(s):  
B. Surendra Nath ◽  
W. Hassan ◽  
S. Nageswara Rao ◽  
N. Vijaya Prakash ◽  
S. Gupta ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Type Species ◽  
Rapd Markers ◽  
Randomly Amplified Polymorphic Dna ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
Random Amplification ◽  
Major Cluster ◽  
Sources Of Infection ◽  
Polymerase Chain

AbstractRandom amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was carried out to assess the genetic diversity of five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworms. A type species, NIK-1s_mys was used as control for comparison. Differences in the spore shape, length and width were observed. Of the 30 decamer random primers tested, 22 primers gave repeatable RAPD profiles and yielded a total of 143 fragments, of which 78 were polymorphic (55%). The resulting data was used to derive genetic similarity values for constructing a dendrogram. The neighbour joining method based on Dice coefficients indicate a major cluster comprising NIK-1s_mys, NIWB-11bp and NIWB-12n, whereas NIWB-13md, NIWB-14b and NIWB-15mb appear to be different from each other as well from the major cluster mentioned above which includes the type species (NIK-1s_mys). Based on the reproducibility of RAPD profiles, we are able to identify these microsporidians as different isolates. The RAPD technique may be useful in detecting sources of infection of this economically important domestic insect.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

scholarly journals Genetic variation and molecular relationships taxa of Conringia heist. ex Fabr. (Brassicaceae) based on RAPD markers in Turkey

Genetika ◽  
2020 ◽  
Vol 52 (1) ◽  
pp. 107-114
Author(s):  
Emre Sevġndġk ◽  
Yavuz Paksoy ◽  
Melike Aydoğan ◽  
Feyzanur Topseçer
Keyword(s):  
Genetic Variation ◽  
Phylogenetic Tree ◽  
Rapd Markers ◽  
Uv Light ◽  
Genetic Relationships ◽  
Genetic Distances ◽  
Genomic Dna Isolation ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Molecular Relationships

In this study, genetic variation and phylogenetic analysis of 13 populations of 6 species belonging to Conringia genus spreading in Turkey were performed using RAPD markers. Genomic DNA isolation from the leaves of the Conringia plant samples was performed via using a commercial kit. Seven RAPD primers were used to identify the genetic diversity between the populations. Polymerase Chain Reaction (PCR) was performed using DNA samples and primers. PCR products were resolved using agarose gel electrophoresis and visualized under UV light. All gel images were analyzed, and the absence and presence of polymorphic bands were scored. The total of 34 DNA bands were detected by seven RAPD primers. PAUP 4.0b10 analysis program was used to calculate phylogenetic tree and genetic distances between the species. The phylogenetic tree was obtained using the UPGMA algorithm and it was composed of two clades. According to the PAUP analysis, the species having the closest distance between each other are C. planisiliqua (Ankara-Aya?) and C. planisiliqua (Ankara-Nall?han) with the value of 0.000 and those having the longest distance are C. grandiflora (Akseki ?ukurk?y) and C. orientalis (Elaz??-Baskil) with the value of 0.6000. The results suggest that the RAPD markers are useful tools to demonstrate the genetic relationships between populations of the Conringia species.

scholarly journals Identification of the B, Q, and native Brazilian biotypes of the Bemisia tabaci species complex using Scar markers

2016 ◽  
Vol 51 (5) ◽  
pp. 555-562 ◽  
Author(s):  
Paulo Roberto Queiroz ◽  
Erica Soares Martins ◽  
Nazaré Klautau ◽  
Luzia Lima ◽  
Lilian Praça ◽  
...  
Keyword(s):  
Bemisia Tabaci ◽  
Species Complex ◽  
Scar Marker ◽  
Random Amplified Polymorphic Dna ◽  
Scar Markers ◽  
Sequence Characterized Amplified Region ◽  
Field Samples ◽  
Scar Primer ◽  
Q Biotype ◽  
B Biotype

Abstract: The objective of this work was to develop sequence-characterized amplified region (Scar) markers to identify the B, Q, and native Brazilian biotypes of the sweet potato whitefly [Bemisia tabaci (Hemiptera: Aleyrodidae)]. Random amplified polymorphic DNA (RAPD) amplification products, exclusive to the B and Brazilian biotypes, were selected after the analysis of 12,000 samples, in order to design a specific Scar primer set. The BT-B1 and BT-B3 Scar markers, used to detect the B biotype, produced PCR fragments of 850 and 582 bp, respectively. The BT-BR1 Scar marker, used to identify the Brazilian biotype, produced a PCR fragment of 700 bp. The Scar markers were tested against the Q biotype, and a flowchart was proposed to indicate the decision steps to use these primers, in order to correctly discriminate the biotypes. This procedure allowed to identify the biotypes that occur in field samples, such as the B biotype. The used set of primers allowed to discriminate the B, Q, and native Brazilian biotypes of B. tabaci. These primers can be successfully used to identify the B biotype of B. tabaci from field samples, showing only one specific biotype present in all cultures.

scholarly journals Use of Random PCR (RAPD) technology to analyze systematic relationships in terrestrial bladderworts (Utricularia L.)

1970 ◽  
Vol 39 (1) ◽  
pp. 97-102 ◽  
Author(s):  
Md Oliur Rahman
Keyword(s):  
Cluster Analysis ◽  
Rapd Markers ◽  
Molecular Taxonomy ◽  
Group Method ◽  
Banding Patterns ◽  
Molecular Approaches ◽  
Pair Group ◽  
Rapd Pcr ◽  
Systematic Relationships ◽  
Polymerase Chain

Polymerase Chain Reaction (PCR) based technology RAPD was tested for its applicability and suitability for molecular systematics of terrestrial bladderworts. PCR was carried out and the resultant products were subjected to agarose gel electrophoresis. The banding patterns were compared among ten species of Utricularia. Out of 40 random oligonucleotide primers examined, 16 primers generated distinguishable bands. Cluster analysis, unweighted pair group method with arithmetic mean (UPGMA) and ordination approach Scatter diagram using Matrix plot were performed based on RAPD fingerprints. The UPGMA and ordination analysis revealed a clear portrayal of systematic relationships in bladderwort species which were concordant with previous studies based on morphology and molecular approaches, indicating the reliability of RAPD markers for estimation of genetic variation and species relationships in Utricularia. Key words: Utricularia; RAPD; PCR; Cluster analysis; Molecular taxonomy DOI: 10.3329/bjb.v39i1.5532Bangladesh J. Bot. 39(1): 97-102, 2010 (June)

Sign in / Sign up

Export Citation Format

Share Document