EFFICIENT ALGORITHMS FOR DEGENERATE PRIMER SEARCH

2007 ◽  
Vol 18 (04) ◽  
pp. 899-910 ◽  
Author(s):  
SUDHA BALLA ◽  
SANGUTHEVAR RAJASEKARAN ◽  
ION I. MANDOIU
Keyword(s):  
Dna Sequences ◽  
Multiplex Polymerase Chain Reaction ◽  
Efficient Algorithms ◽  
Degenerate Primer ◽  
Experimental Comparison ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Np Complete ◽  
Polymerase Chain ◽  
Input Strings

Degenerate primers are used to amplify a given set of genomic sequences using a technique called Multiplex Polymerase Chain Reaction (MP-PCR). The problem of minimizing the number of degenerate primers required to amplify a given set of DNA sequences, also known as the Degenerate Primer Design Problem (DPDP), has been extensively studied in the literature and proven to be NP-Complete. In this paper we present efficient algorithms for solving DPDP. For example, one of the algorithms we give in this paper is iterative and has a runtime of O(b|Σ|log|Σ|dn2mp) to select a set of p degenerate primers, each of given length l and degeneracy at most d, for n sequences each of length m in the input, the number of candidates retained in each iteration being b. Σ is the alphabet of the input strings. This is an improvement over the runtime of the best known prior algorithm, MIPS by Souvenir et al. [15], which has a runtime of O(bn3mp). We provide an experimental comparison of MIPS and our algorithms.

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

2019 ◽  
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 (Green et al. 2015). 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 Deconstructing the Polymerase Chain Reaction II: An improved workflow and effects on artifact formation and primer degeneracy

2019 ◽  
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 (Green et al. 2015). 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.

Detection of alpha-thalassemias by multiplex polymerase chain reaction

1994 ◽  
Vol 40 (12) ◽  
pp. 2260-2266 ◽  
Author(s):  
L J Bowie ◽  
P L Reddy ◽  
M Nagabhushan ◽  
P Sevigny
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Multiplex Polymerase Chain Reaction ◽  
Clinical Laboratory ◽  
Laboratory Method ◽  
Control Sequence ◽  
Chain Reaction ◽  
Simple Method ◽  
Alpha Thalassemia ◽  
Polymerase Chain

Abstract Although alpha-thalassemia is the most common genetic abnormality in the world, there is currently no routine laboratory method to definitively identify individuals who are affected. We describe a rapid and simple method that utilizes deletion-sensitive primers to amplify normal DNA sequences. Deletions involving the regions responsible for most of the alpha-thalassemia cases in the US prevent amplification with these primers. In tests with DNA isolated from small amounts (10 microL) of whole blood, the deletion-sensitive primers gave rise to the expected 248- and 375-bp (base pair) amplification products in normal individuals. These primers, along with primers designed to bind to a nonaffected control sequence from the hemoglobin beta chain, could be amplified simultaneously (multiplex polymerase chain reaction). This made it possible to detect heterozygotes for alpha-thalassemia-2 (one alpha locus deleted) by determining the ratios of the 248- and 375-bp amplification products to the product of the control sequence (268 bp). The method is rapid and simple and can be performed in a routine clinical laboratory.

Virus respiratoires dans les pneumonies associées aux soins

2018 ◽  
Vol 27 (3) ◽  
pp. 217-227
Author(s):  
P. Loubet ◽  
G. Voiriot ◽  
M. Neuville ◽  
B. Visseaux ◽  
J.-F. Timsit
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Biologie Moléculaire ◽  
Mise En Œuvre ◽  
Polymerase Chain ◽  
Infection Bactérienne

Les pneumonies acquises à l’hôpital (PAH) sont fréquentes. À l’ère des techniques diagnostiques de biologie moléculaire (multiplex polymerase chain reaction), les rares données disponibles estiment que les virus respiratoires sont impliqués dans 22 à 32 % des épisodes. Les patients immunodéprimés constituent probablement la population la plus à risque. La présentation clinique et radiologique ne diffère pas entre pneumonies bactériennes, virales et mixtes (virus–bactérie). L’excrétion prolongée de virus respiratoires dans les voies aériennes a été rapportée chez les patients immunodéprimés. Elle pourrait promouvoir la co-infection bactérienne, associée à des durées d’hospitalisation prolongées. L’acquisition intrahospitalière a été démontrée chez tous les virus respiratoires. Elle encourage la mise en œuvre et le respect des mesures d’hygiène et de confinement, dans l’objectif de protéger soignants, visiteurs et patients. De nombreux points restent largement méconnus, relatifs aux interactions entre virus respiratoires et pathogènes non viraux, aux périodes d’incubation, ou encore aux durées d’excrétion virale. L’amélioration des techniques diagnostiques et l’accumulation de données épidémiologiques et cliniques devraient permettre de mieux appréhender le rôle des virus respiratoires dans les PAH. Cette meilleure connaissance aidera à rationaliser l’utilisation des tests de détection et facilitera l’interprétation de leurs résultats. Elle guidera aussi le clinicien dans l’utilisation future des nombreuses molécules antivirales actuellement en développement clinique chez l’homme.

Molecular Analysis of Uropathogenic E.coli Isolates from Urinary Tract Infections

2019 ◽  
Vol 19 (3) ◽  
pp. 322-326 ◽  
Author(s):  
Hassan Valadbeigi ◽  
Elham Esmaeeli ◽  
Sobhan Ghafourian ◽  
Abbas Maleki ◽  
Nourkhoda Sadeghifard
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Multiplex Polymerase Chain Reaction ◽  
Virulence Genes ◽  
Uropathogenic Escherichia Coli ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tract Infections

Introduction: The aim of the current study was to investigate the prevalence of virulence genes in uropathogenic Escherichia coli (UPEC) isolates in Ilam. Materials and Methods: For this purpose, a total of 80 UPEC isolates were collected for patients with UTIs during a 6 months period. The multiplex polymerase chain reaction (multiplex PCR) was used to detect the papEF, fimH, iucD, hlyA, fyuA, and ompT genes. Results: The prevalence of fimH, papEF, iucD, fyuA, hlyA, hlyA, and ompT genes were 87.5%, 47.5%, 60%, 67.5%, 27.5%, 47.5% and 71.2%, respectively. Among all of the isolates, 27 profiles were obtained. Conclusion: Our findings demonstrated that the most prevalence was found for fimH, and different distribution of virulence genes suggested different ability of pathogenicity.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

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