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.

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.

scholarly journals Evaluation of quantitative polymerase chain reaction to assessnosZgene prevalence in mixed microbial communities

2007 ◽  
Vol 53 (5) ◽  
pp. 636-642 ◽  
Author(s):  
Steven D. Siciliano ◽  
Wai Ma ◽  
Shane Powell
Keyword(s):  
Polymerase Chain Reaction ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Ralstonia Eutropha ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Total Community ◽  
Polymerase Chain ◽  
Template Dna

The usefulness of quantitative polymerase chain reaction (QPCR) to measure nosZ gene prevalence in a multi-template reaction was assessed by comparing 19 nosZ template DNA samples and 91 model communities. Efficiencies of the QPCR varied but were not significantly different among nosZ genotypes and were not linked to genetic distance from Ralstonia eutropha . nosZ genotype QPCR efficiencies obtained from isolated denitrifiers were higher (84.8%) than those obtained from excised denaturing gradient gel electrophoresis bands or clones of PCR products from total community DNA (ca. 60%). Analysis of the model communities indicated that QPCR accurately predicts gene prevalence in communities composed of up to six templates.

A polymerase chain reaction method for detecting dwarf mistletoe infection in Douglas-fir and western larch

1999 ◽  
Vol 29 (9) ◽  
pp. 1317-1321 ◽  
Author(s):  
M Marler ◽  
D Pedersen ◽  
T Mitchell-Olds ◽  
R M Callaway
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Method ◽  
Positive Control ◽  
Rbcl Gene ◽  
Larix Occidentalis ◽  
Chain Reaction ◽  
Dwarf Mistletoe ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Template Dna

Early detection and management of dwarf mistletoe (Arceuthobium spp.) is currently limited by the inability to rapidly detect infection during the 2- to 5-year endophyte phase of the parasite. We describe a polymerase chain reaction (PCR) technique for detecting Arceuthobium douglasii Engelm. and Arceuthobium laricis Engelm. in tissues of its hosts, Pseudotsuga menziesii (Mirb.) Franco and Larix occidentalis Nutt. DNA was extracted from branches of 15 infected and 15 uninfected P. menziesii. The PCR product amplified by using the Arceuthobium specific primer in the rbcL gene from Arceuthobium template DNA was a fragment of 708 pairs of bases in length. This product was amplified from all branches that were visibly infected, but the fragment was not generated from any samples known to be uninfected. The PCR product from conifer DNA was a fragment of 385 pairs of bases in length and was not amplified from pure mistletoe DNA; this was amplified as an internal positive control. The primers developed for P. menziesii and A. douglasii also worked on L. occidentalis and A. laricis. This method detected mistletoe DNA in 7 of 29 P. menziesii branches and 3 of 21 L. occidentalis branches that did not have external symptoms of infection and are presumed to be the result of the endophyte phase. This method provides a useful tool for experimental applications and for managing the spread of dwarf mistletoe.

Analysis of the inhibition of nucleic acid dyes on polymerase chain reaction by capillary electrophoresis

2016 ◽  
Vol 8 (11) ◽  
pp. 2330-2334 ◽  
Author(s):  
Zhenqing Li ◽  
Chenchen Liu ◽  
Siyao Ma ◽  
Dawei Zhang ◽  
Yoshinori Yamaguchi
Keyword(s):  
Polymerase Chain Reaction ◽  
Capillary Electrophoresis ◽  
Nucleic Acid ◽  
Acid Dyes ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Accurate Quantification

An integrated polymerase chain reaction (PCR) and capillary electrophoresis (CE) system can realize accurate quantification of the target PCR product by adding labeling dyes to the PCR reagents, because CE can discriminate all the subsequent nucleic acids, including the primers, non-specific and specific PCR products.

Triple-Helical Capture Assay for Quantification of Polymerase Chain Reaction Products

1992 ◽  
Vol 38 (5) ◽  
pp. 687-694 ◽  
Author(s):  
C P Vary
Keyword(s):  
Polymerase Chain Reaction ◽  
Fluorescence Analysis ◽  
Automated Analysis ◽  
Pcr Primers ◽  
Reaction Products ◽  
Chain Reaction ◽  
Capture Assay ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract This method for rapid, automated analysis of polymerase chain reaction (PCR) products makes use of PCR primers containing 5'-polypyrimidine sequences. Polypyrimidine-"headed" primers confer to the PCR product the ability to form triple helical complexes with a third polypyrimidine oligonucleotide. Third-strand oligonucleotides are modified to serve as either capture reagents or detection reagents for PCR products. Automated quantitative measurement of the PCR product is achieved by using latex bead-based fluorescence analysis. The use of triple-instead of double-helical interactions avoids the usual requirements of complex blocking reagents, time- and labor-intensive washing steps, and long times for color development. The method also provides rapid, sequence-specific capture and detection of PCR products without the need to denature the double-stranded PCR product. The assay is demonstrated with use of both PCR primer-derived and endogenous triple-helix-forming sequences resulting from PCR of several bacterial and viral target nucleic acids.

scholarly journals A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Oleg S. Alexandrov ◽  
Olga V. Razumova ◽  
Gennady I. Karlov
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Markers ◽  
Dna Markers ◽  
5S Rdna ◽  
Chain Reaction ◽  
Closely Related Species ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Species Specific ◽  
Shepherdia Canadensis

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95–96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

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