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

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.

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

Clinical Chemistry ◽

10.1093/clinchem/39.9.1927 ◽

1993 ◽

Vol 39 (9) ◽

pp. 1927-1933 ◽

Cited By ~ 39

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.

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Multiplex-Polymerase Chain Reaction Assay for the Authentication of the Mackerel Scomber colias in Commercial Canned Products

Journal of AOAC International ◽

10.1093/jaoac/89.3.708 ◽

2006 ◽

Vol 89 (3) ◽

pp. 708-711 ◽

Cited By ~ 3

Author(s):

Carlos Infante ◽

Manuel Manchado

Keyword(s):

Polymerase Chain Reaction ◽

Multiplex Polymerase Chain Reaction ◽

Rrna Gene ◽

Chain Reaction ◽

Nontranscribed Spacer ◽

Specific Pcr ◽

Scomber Colias ◽

Pcr Product ◽

Polymerase Chain ◽

Specific Fragment

Abstract A multiplex-polymerase chain reaction (PCR) system was developed for the authentication of the mackerel Scomber colias in commercial canned products. This novel method consists of an S. colias-specific fragment [159 base pairs (bp)] located in the nontranscribed spacer (NTS) sequence, and a Scomber genus-specific PCR product in the 5S rRNA gene (196201 bp) as a positive amplification control. The system was assayed using 18 different canned products labeled as S. colias. A positive identification was made in all but one sample, revealing this methodology as a potential molecular tool for direct application in the authentication of S. colias canned products.

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Capillary Electrophoresis of Short Tandem Repeat (STR) Polymerase Chain Reaction (PCR) Products from a Commercial Multiplex Kit

Forensic DNA Biology ◽

10.1016/b978-0-12-394585-3.00012-2 ◽

2013 ◽

pp. 105-113

Author(s):

Kelly M. Elkins

Keyword(s):

Polymerase Chain Reaction ◽

Capillary Electrophoresis ◽

Tandem Repeat ◽

Short Tandem Repeat ◽

Chain Reaction ◽

Pcr Products ◽

Polymerase Chain ◽

Short Tandem

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Multiplex Polymerase Chain Reaction Method for Detection of Bovine Materials in Foodstuffs

Journal of AOAC International ◽

10.1093/jaoac/86.4.764 ◽

2003 ◽

Vol 86 (4) ◽

pp. 764-767 ◽

Cited By ~ 7

Author(s):

Hong-Wei Gao ◽

Da-Bing Zhang ◽

Ai-Hu Pan ◽

Wan-Qi Liang ◽

Cheng-Zhu Liang

Keyword(s):

Polymerase Chain Reaction ◽

Genomic Dna ◽

Polymerase Chain Reaction Method ◽

Rapid Identification ◽

Ribosomal Gene ◽

Effective Control ◽

Chain Reaction ◽

Specific Pcr ◽

Pcr Product ◽

Polymerase Chain

Abstract Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained >0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.

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

F1000Research ◽

10.12688/f1000research.5813.1 ◽

2014 ◽

Vol 3 ◽

pp. 320 ◽

Cited By ~ 1

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.

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

Clinical Chemistry ◽

10.1093/clinchem/38.5.687 ◽

1992 ◽

Vol 38 (5) ◽

pp. 687-694 ◽

Cited By ~ 12

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.

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Faculty Opinions recommendation of One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1012550.186179 ◽

2003 ◽

Author(s):

Richard L Stevens

Keyword(s):

Polymerase Chain Reaction ◽

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Point Mutations ◽

Chain Reaction ◽

Step Detection ◽

Hybridization Probes ◽

One Step ◽

Polymerase Chain

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

Plants ◽

10.3390/plants10010004 ◽

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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Use of the polymerase chain reaction for detection of Fusarium graminearum in bulgur wheat

Food Science and Technology ◽

10.1590/s0101-20612012005000027 ◽

2012 ◽

Vol 32 (1) ◽

pp. 201-208 ◽

Cited By ~ 1

Author(s):

Carla Bertechini Faria ◽

Giovana Caputo Almeida-Ferreira ◽

Karina Bertechine Gagliardi ◽

Tatiane Cristina Albuquerque Alves ◽

Dauri José Tessmann ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Fusarium Graminearum ◽

Genomic Dna ◽

Solid Medium ◽

Food Contamination ◽

Chain Reaction ◽

Specific Pcr ◽

Culture Characteristics ◽

Mycotoxigenic Fungi ◽

Polymerase Chain

The detection of mycotoxigenic fungi in foodstuff is important because their presence may indicate the possible associated mycotoxin contamination. Fusarium graminearum is a wheat pathogen and a producer of micotoxins. The polymerase chain reaction (PCR) has been employed for the specific identification of F. graminearum. However, this methodology has not been commonly used for detection of F. graminearum in food. Thus, the objective of the present study was to develop a molecular methodology to detect F. graminearum in commercial samples of bulgur wheat. Two methods were tested. In the first method, a sample of this cereal was contaminated with F. graminearum mycelia. The genomic DNA was extracted from this mixture and used in a F. graminearum specific PCR reaction. The F. graminearum species was detected only in samples that were heavily contaminated. In the second method, samples of bulgur wheat were inoculated on a solid medium, and isolates having F. graminearum culture characteristics were obtained. The DNA extracted from these isolates was tested in F. graminearum specific PCR reactions. An isolate obtained had its trichothecene genotype identified by PCR. The established methodology could be used in surveys of food contamination with F. graminearum.

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