Ligation mediated PCR performed at low denaturation temperatures--PCR melting profiles

Masny, Aleksander; Płucienniczak, Andrzej

doi:10.1093/nar/gng116

Cited by 45 publications

(65 citation statements)

References 11 publications

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“…Recently, the PCR melting profile (PCR MP) method appeared to be very useful in genetic diversity studies of several bacterial genera, mainly Enterobacteriaceae (Masny and Płucienniczak 2003;Krawczyk et al 2006Krawczyk et al , 2007. This method consists of three steps: digestion with restriction enzyme, ligation of oligonucleotide adapters corresponding to the restriction sites, and PCR amplification of the ligation products using low denaturation temperatures T d (80-88°C).…”

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“…Lowering of T d decreases the number of amplified fragments and leads to limited and specific amplification of less stable DNA fragments. This DNA fingerprinting method allows differentiation of bacteria, even at strain level, based on heterogeneity of their GC content and length of genomic DNA fragments obtained after digestion with endonucleases (Masny and Płucienniczak 2003).…”

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The use of PCR melting profile for typing of Pseudomonas syringae isolates from stone fruit trees

2009

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Of thirty fluorescent Pseudomonas isolates originating from symptomatic tissues of sweet (Prunus avium) and sour cherry (Prunus cerasus), plum (Prunus domestica), peach (Prunus persica) and apricot (Prunus armeniaca), 23 were identified as P. syringae using LOPAT tests. Further characterization of those isolates by GATTa and L-lactate utilization tests showed that 10 of them belonged to race 1, six to race 2 of P. syringae pv. morsprunorum

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The use of PCR melting profile for typing of Pseudomonas syringae isolates from stone fruit trees

2009

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“…Here, we show application of a modified PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR), developed by Masny and Płucienniczak (9), for epidemiological studies. The proposed method allows gradual amplification of the genomic DNA differing in the thermal stability starting from the less stable DNA fragments amplified at lower denaturation temperature (T d ) values to more stable ones amplified at higher T d values.…”

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Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation

et al. 2006

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In search of an effective DNA typing technique for hospital epidemiology use, the performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR) of bacterial DNA was tested. A number of Escherichia coli isolates from patients of the Clinical Hospital in Gdańsk, Poland, were examined. We found that the PCR MP technique is a rapid method that offers good discriminatory power and excellent reproducibility and may be applied for epidemiological studies. The usefulness of the PCR MP for molecular typing was compared with the pulsedfield gel electrophoresis method, which is currently considered the gold standard for epidemiological studies of isolates recovered from patients and the environment. Clustering of PCR MP fingerprinting data matched pulsed-field gel electrophoresis data. The features of the PCR MP technique are discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning E. coli that may occur in a hospital.Over the past 20 years, a significant number of DNA-based techniques have been introduced into the field of bacterial characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. DNA fingerprinting involves the display of a set of DNA fragments from a specific DNA sample. A variety of DNA fingerprinting techniques are presently available (1-7, 11-13), most of which use PCR for detection of fragments. The choice of fingerprinting technique depends on the type of application. Ideally, a fingerprinting technique should require no prior investments in terms of sequence analysis, primer synthesis, or characterization of DNA probes. A number of fingerprinting methods which meet these requirements have been developed. The fingerprints are obtained by visualizing many parts of the genome. Differences in these fingerprints between individuals are interpreted as genetic distances. Obviously, the differences should reflect variations in DNA rather than artifacts due to a nonrobust method. Furthermore, the method should provide the appropriate level of discriminatory power, and it should be relatively rapid and cheap, especially in large-scale population genetic studies. Macrorestriction analysis of genomic DNA followed by pulsed-field gel electrophoresis (REA-PFGE) has become the "gold standard" for molecular typing (10). In recent years, some alternative techniques have been successfully applied for the typing of bacteria below the species level. These include amplification-based methods, such as amplified fragment length polymorphisms (11), random amplification of polymorphic DNA (3, 12, 13), and amplification of DNA fragments surrounding rare restriction sites (5, 6, 8). These techniques are now applied more and more because they involve less time, comparably low cos...

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“…The enzyme attaches to this mutagenic duplex and continues the synthesis of the new strand, which is one repeat shorter. At a temperature about 107C lower for the PCR denaturation step, not only will a substantial fraction of the DNA template remain double-stranded [54,55], but a fraction of template strands will also not dissociate from the products of incomplete new strand synthesis. Thus, the opportunity of their misalignment upon the reassociation of separated strands will be reduced.…”

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Genotyping of simple sequence repeat factors implicated in shadow band generation revisited

Olejniczak

Krzyżosiak

2006

Electrophoresis

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PCR amplification of microsatellite sequences generates, besides the main product corresponding to allele size, also additional, undesired products usually shorter by multiples of the repeated unit. These extra products known as shadow bands or stutter products may complicate genotyping. The mechanism by which these artifacts are formed is not well understood and so no effective remedy has been found to cope with these spurious products. In this study, using the DNA templates containing the CAG/CTG repeats flanked by gene-specific sequences and universal priming sites, we analyzed the effects of many PCR variables on the shadow band generation. The most important result was that at the decreased temperature of the denaturation step during PCR cycling the shadow bands were either not formed or were strongly suppressed. Several possible sources of this effect are discussed.

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Ligation mediated PCR performed at low denaturation temperatures--PCR melting profiles

Cited by 45 publications

References 11 publications

The use of PCR melting profile for typing of Pseudomonas syringae isolates from stone fruit trees

The use of PCR melting profile for typing of Pseudomonas syringae isolates from stone fruit trees

Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation

Genotyping of simple sequence repeat factors implicated in shadow band generation revisited

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