The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×106 reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.

/pdf/reducing-the-effects-of-pcr-amplification-and-sequencing-4zw8t09xbt.pdf

Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies

Virtually all empirical ecological studies require species identification during data collection. DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA (soil, water, faeces, etc.). It can be implemented for both modern and ancient environmental samples. The availability of next-generation sequencing platforms and the ecologists' need for high-throughput taxon identification have facilitated the emergence of DNA metabarcoding. The potential power of DNA metabarcoding as it is implemented today is limited mainly by its dependency on PCR and by the considerable investment needed to build comprehensive taxonomic reference libraries. Further developments associated with the impressive progress in DNA sequencing will eliminate the currently required DNA amplification step, and comprehensive taxonomic reference libraries composed of whole organellar genomes and repetitive ribosomal nuclear DNA can be built based on the well-curated DNA extract collections maintained by standardized barcoding initiatives. The near-term future of DNA metabarcoding has an enormous potential to boost data acquisition in biodiversity research.

Towards next‐generation biodiversity assessment using DNA metabarcoding

Developments in industrially important thermostable enzymes: a review.

DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, and potential applications.

/pdf/pyrosequencing-sheds-light-on-dna-sequencing-4vpt46skyo.pdf

Pyrosequencing sheds light on DNA sequencing.

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 · 10 ‐6 ) < Deep Vent (2.7 · 10 ‐6 ) < Vent (2.8 · 10 ‐6 ) < Taq (8.0 · 10 ‐6 ) << exo ‐ Pfu and UlTma (~5 · 10 ‐5 ). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2‐3 mM MgSO4 and 100‐300 μM each dNTP and at pH 8.5‐9.1. Under these conditions, the error rate of exo ‐ Pfu was ~40-fold higher (5 · 10 ‐5 ) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased ~2-fold, while the error rate of exo ‐ Pfu increased ~9-fold. An increase in error rate with pH has also been noted for the exonucleasedeficient DNA polymerases Taq and exo ‐ Klenow, suggesting that the parameters which influence replication error rates may be similar in pol I- and α-like polymerases. Finally, the fidelity of ‘long PCR’ DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but ~3‐4-fold higher than the error rate of Pfu DNA polymerase.

/pdf/pcr-fidelity-of-pfu-dna-polymerase-and-other-thermostable-33w1uh1kgi.pdf

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

https://www.gene-quantification.de/arezi-2003.pdf

Amplification efficiency of thermostable DNA polymerases.

We discovered a thermostable enzyme from the archaeon Pyrococcus furiosus (Pfu), which increases yields of PCR product amplified with Pfu DNA polymerase. A high molecular mass (>250 kDa) complex with PCR-enhancing activity was purified from Pfu extracts. The complex is a multimer of two discrete proteins, P45 and P50, with significant similarity to bacterial dCTP deaminase/dUTPase and DNA flavoprotein, respectively. When tested in PCR, only recombinant P45 exhibited enhancing activity. P45 was shown to function as a dUTPase, converting dUTP to dUMP and inorganic pyrophosphate. Pfu dUTPase improves the yield of products amplified with Pfu DNA polymerase by preventing dUTP incorporation and subsequent inhibition of the polymerase by dU-containing DNA. dUTP was found to accumulate during PCR through dCTP deamination and to limit the efficiency of PCRs carried out with archaeal DNA polymerases. In the absence of dUTP inhibition, the combination of cloned Pfu DNA polymerase and Pfu dUTPase (PfuTurbo DNA polymerase) can amplify longer targets in higher yield than Taq DNA polymerase. In vivo, archaeal dUTPases may play an essential role in preventing dUTP incorporation and inhibition of DNA synthesis by family B DNA polymerases.

https://www.pnas.org/content/pnas/99/2/596.full.pdf

Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation

The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

Holly H. Hogrefe

Papers

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

Amplification efficiency of thermostable DNA polymerases.

Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation

Compositions and methods utilizing DNA polymerases

A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage.