Efficacy of SSH PCR in isolating differentially expressed genes

Background  

Suppression Subtractive Hybridization PCR (SSH PCR) is a sophisticated cDNA subtraction method to enrich and isolate differentially expressed genes. Despite its popularity, the method has not been thoroughly studied for its practical efficacy and potential limitations.     

Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

Background  

Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.     

A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step

Background  

Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.     

Long‐range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Aims

An extra‐long‐range quantitative PCR (LR‐qPCR) method was developed for estimating genome damage to adenovirus 2 caused by UV irradiation. The objective was to use LR‐qPCR as a rapid method to determine adenovirus UV inactivation.

Methods

The LR‐qPCR consisted of two steps: a long‐range PCR (up to 10 kb fragment) and a real‐time, quantitative (q) PCR for quantifying the products of the first PCR. We evaluated LR‐qPCR with adenovirus irradiated with medium‐pressure (MP, polychromatic emission) and low‐pressure (LP, 254 nm) mercury vapour lamps and compared results with cell culture infectivity.

Results

Using LR‐qPCR, a fragment of 6 kb estimated DNA damage in a linear relationship to doses between 0 and 20 mJ cm^?2, and a 1‐kb fragment related linearly to doses between 20 and 100 mJ cm^?2. The LR‐qPCR results for the 6‐kb fragment were similar to infectivity assays results for adenovirus exposed to MP UV. For adenovirus irradiated with LP lamps, LR‐qPCR results for the shorter fragment size (1 kb) were similar to reduction in viral infectivity. No difference was observed between 10 and 6 kb LR‐qPCR results.

Conclusion

The LR‐qPCR can be used as a tool for estimating DNA damage caused by UV in adenovirus. The LR‐qPCR results were related to reduction in viral infectivity.

Significance and Impact of the Study

The use of LR‐qPCR to determine DNA damage and estimate inactivation of adenovirus 2 from UV disinfection allows for same‐day results compared with >7 days required for cell culture. This accelerates adenovirus inactivation results for the water industry where adenovirus is used as a representative virus for crediting UV systems. This PCR approach provides a framework that can be used for other viral viability assays using the inhibition of amplification of viral nucleic acid after pretreatments, such as propidium monoazide, and for cellular biology studies of DNA damage.     

A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering

Background  

The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR.     

Shape based kinetic outlier detection in real-time PCR

Background  

Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD.     

Revealing biases inherent in recombination protocols

Background  

The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.     

Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma

Background  

Epstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR).     

A standard curve based method for relative real time PCR data processing

Background  

Currently real time PCR is the most precise method by which to measure gene expression. The method generates a large amount of raw numerical data and processing may notably influence final results. The data processing is based either on standard curves or on PCR efficiency assessment. At the moment, the PCR efficiency approach is preferred in relative PCR whilst the standard curve is often used for absolute PCR. However, there are no barriers to employ standard curves for relative PCR. This article provides an implementation of the standard curve method and discusses its advantages and limitations in relative real time PCR.     

A high-throughput pipeline for the design of real-time PCR signatures

Background  

Pathogen diagnostic assays based on polymerase chain reaction (PCR) technology provide high sensitivity and specificity. However, the design of these diagnostic assays is computationally intensive, requiring high-throughput methods to identify unique PCR signatures in the presence of an ever increasing availability of sequenced genomes.     

Product category rules alignment workshop, October 4, 2011 in Chicago, IL, USA

Purpose  

A workshop on Product Category Rule (PCR) alignment was organized by the American Center for LCA PCR Committee. PCR alignment refers to the process of assuring that PCRs (rules for developing LCA-based claims like EPDs) developed by different parties are consistent within product categories.     

Detection of Helicobacter pylori in stool samples of young children using real‐time polymerase chain reaction

Background

The aims of this study were to develop and validate a multiplex real‐time polymerase chain reaction (q‐PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori‐positive samples.

Materials and methods

Archived stool samples from 188 children aged 6‐9 years and 272 samples of 92 infants aged 2‐18 months were tested for H. pylori antigens using enzyme immunoassay (EIA). A multiplex q‐PCR assay was designed to detect H. pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q‐PCR and EIA.

Results

Laboratory validation of the q‐PCR assay using quantitated H. pylori ATCC 43504 extracted DNA showed S‐shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross‐reactivity with other bacterial pathogens was noted. Applying the multiplex q‐PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H. pylori infection was 50% (95% CI 43%‐57%) by q‐PCR (urease cycle threshold <44) vs 59% (95% CI 52%‐66%) by EIA. Kappa coefficient was .80 (P < .001) and .44 (P < .001) for children aged 6‐9 years and 2‐18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing.

Conclusions

The developed q‐PCR can be used as a cotechnique to enhance the accuracy of H. pylori detection in epidemiological studies and in clinical settings.     

Effects of polymerase,template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method

Background  

The primer and amplicon length have been found to affect PCR based estimates of microbial diversity by pyrosequencing, while other PCR conditions have not been addressed using any deep sequencing method. The present study determined the effects of polymerase, template dilution and PCR cycle number using the Solexa platform.     

Multiplex real‐time PCR assays for detection of four seedborne spinach pathogens

Aims

To develop multiplex TaqMan real‐time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach.

Methods and Results

Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real‐time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real‐time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed.

Conclusions

The real‐time PCR assays were species‐specific and sensitive. Singular or multiplex real‐time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed.

Significance and Impact of the Study

The freeze‐blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real‐time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.     

FCI: an R-based algorithm for evaluating uncertainty of absolute real-time PCR quantification

Background  

FCI is an R code for analyzing data from real-time PCR experiments. This algorithm estimates standard curve features as well as nucleic acid concentrations and confidence intervals according to Fieller's theorem.     

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

Background  

The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls.     

BISMA - Fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences

Background  

Bisulfite sequencing is a popular method to analyze DNA methylation patterns at high resolution. A region of interest is targeted by PCR and about 20-50 subcloned DNA molecules are usually analyzed, to determine the methylation status at single CpG sites and molecule resolution.     

IS<Emphasis Type="Italic">1111</Emphasis> insertion sequences of <Emphasis Type="Italic">Coxiella burnetii</Emphasis>: characterization and use for repetitive element PCR-based differentiation of <Emphasis Type="Italic">Coxiella burnetii</Emphasis> isolates

Background  

Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method.     

Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR

Background  

Robust designs of PCR-based molecular diagnostic assays rely on the discrimination potential of sequence variants affecting primer-to-template annealing. However, for accurate quantitative PCR (qPCR) assessment of gene expression in populations with gene polymorphisms, the effects of sequence variants within primer binding sites must be minimized. This dichotomy in PCR applications prompted us to design experiments to specifically address the quantitative nature of PCR amplifications with oligonucleotides containing mismatches.