Use of degenerate primers and heat-soaked polymerase chain reaction (PCR) to clone a serine protease antigen fromDermatophilus congolensis

1997 ◽  
Vol 75 (5) ◽  
pp. 484-491 ◽  
Author(s):  
Ontiretse M Mine ◽  
Patrick R Carnegie
Keyword(s):  
Polymerase Chain Reaction ◽  
Serine Protease ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain

scholarly journals Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7121
Author(s):  
Ankur Naqib ◽  
Silvana Poggi ◽  
Stefan J. Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Microbial Community ◽  
Annealing Temperature ◽  
Degenerate Primer ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain ◽  
Dna Template ◽  
Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

scholarly journals Light Microscopy and PCR Based Detection of the Causal Agent of Leaf Mosaic of Jute

2013 ◽  
Vol 22 (1-2) ◽  
pp. 19-25
Author(s):  
KMG Dastogeer ◽  
MA Ali ◽  
M Ashrafuzzaman
Keyword(s):  
Polymerase Chain Reaction ◽  
Light Microscope ◽  
Inclusion Bodies ◽  
Causal Agent ◽  
Healthy Plant ◽  
Infected Leaf ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Leaf Tissues ◽  
Polymerase Chain

Leaf mosaic transmitted by whitefly is a devastating disease of jute. It is thought to be caused by a virus belonging to begomovirus genus under geminivirus family. To confirm the identity of the causal agent, infected and healthy leaves were studied using light microscope and by using polymerase chain reaction (PCR) technique of DNA. The inclusion bodies were observed under light microscope as large, blue-violet, prominent inclusion bodies in the nucleus of the infected leaf tissues. In molecular detection technique DNA from infected and healthy plants was extracted and analyzed by polymerase chain reaction (PCR) using degenerate primers PALIv1978/PARIc496. PCR fragment of the expected size 1.2kb for the common region (CR) in the geminivirus were obtained from infected plants. DNA collected from healthy plant did not show any band during electrophoresis. Therefore, it can be concluded that leaf mosaic of jute is cause by a virus.DOI: http://dx.doi.org/10.3329/pa.v22i1-2.16463 Progress. Agric. 22(1 & 2): 19 - 25, 2011

Cloning of Baeyer-Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers

2003 ◽  
Vol 5 (3) ◽  
pp. 174-182 ◽  
Author(s):  
Jan B. van Beilen ◽  
Frederic Mourlane ◽  
Markus A. Seeger ◽  
Jasminka Kovac ◽  
Zhi Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain

scholarly journals Polyvalent Degenerate Oligonucleotides Reverse Transcription-Polymerase Chain Reaction: A Polyvalent Detection and Characterization Tool for Trichoviruses, Capilloviruses, and Foveaviruses

2005 ◽  
Vol 95 (6) ◽  
pp. 617-625 ◽  
Author(s):  
Xavier Foissac ◽  
Laurence Svanella-Dumas ◽  
Pascal Gentit ◽  
Marie-Josée Dulucq ◽  
Armelle Marais ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Unknown Etiology ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Sequence Comparisons ◽  
Pcr Products ◽  
Chlorotic Leaf ◽  
Polymerase Chain

A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

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