Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates

Background While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC \u3e 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds. Findings Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR. Conclusions BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation

Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence

BackgroundPolymerase chain reaction (PCR) is an extremely sensitive method that often demands optimization, especially when difficult templates need to be amplified. The aim of the present study was to optimize the PCR conditions for amplification of the epidermal growth factor receptor (EGFR) promoter sequence featuring an extremely high guanine-cytosine (GC) content in order to detect single nucleotide polymorphisms -216G gt T and -191C gt A. MethodsGenomic DNA used for amplification was extracted from formalin-fixed paraffin-embedded lung tumor tissue and PCR products were detected by agarose gel electrophoresis. ResultsResults showed that addition of 5% dimethyl sulfoxide (DMSO), as well as DNA concentration in PCR reaction of at least 2 g/ml, were necessary for successful amplification. Due to high GC content, optimal annealing temperature was 7 degrees C higher than calculated, while adequate MgCl2 concentration ranged from 1.5 to 2.0 mM. ConclusionIn conclusion, EGFR promoter region is a difficult PCR target, but it could be amplified after optimization of MgCl2 concentration and annealing temperature in the presence of DMSO and the DNA template of acceptable concentration

Planktonic and sediment-associated aerobic methanotrophs in two seep systems along the North American margin

Methane vents are of significant geochemical and ecological importance. Notable progress has been made towards understanding anaerobic methane oxidation in marine sediments, however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs, and distantly related to established methanotrophic clades. Water-column associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment

Desenvolvimento de uma mistura reacional de baixo custo para PCR quantitativa baseada em SYBR® Gold

Orientador: Rafael Nobrega StippTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A PCR em tempo real ou quantitativa (qPCR) é amplamente utilizada para quantificação de DNA e apresenta um alto custo nas pesquisas das áreas biológicas, sobretudo pela utilização de reagentes importados. O objetivo foi desenvolver uma mistura reacional para a qPCR com especificidade, eficiência e sensibilidade equivalente ao produto industrializado importado. Desse modo, foram preparados sistemas tampões que variavam em relação às concentrações de SYBR® Green ou SYBR® Gold, MgCl2 e Taq DNA Polimerase Brazil. Para potencializar a reação, aditivos como: albumina sérica bovina, dimetil sulfóxido, formamida, polietileno glicol 400, Triton x-100, trealose e Tween-20 foram utilizados em variadas concentrações de forma separada ou combinada. O DNA genômico de Porphyromonas gingivalis (300 a 0,003 ng) e primers espécie-específico foram utilizados para a comparação inicial entre as misturas. Para garantir a aplicabilidade, as misturas com melhores resultados foram avaliadas contra DNA de outras espécies: Streptococcus sanguinis, Mus Musculus e Candida albicans. Além disso, foram submetidas à testes de estabilidade. As misturas foram equiparadas ao reagente comercial Power SYBR Green qPCR Master Mix Kit© através dos valores de Cycle threshold e das curvas de melting. A mistura com melhor eficiência, especificidade e estabilidade foi chamada de Master Mix para qPCR não comercial. A eficiência do Master Mix para qPCR não comercial foi até 28% superior ao produto comercial, variando de acordo com a concentração, origem e qualidade do material genético. O preço atual do consumível da reação de qPCR pode ser reduzido em até 70% empregando a mistura reacional proposta. Em conclusão, foi desenvolvida uma mistura reacional para PCR, com alta eficiência e especificidade, e um baixo custo. A mistura permite alterações individuais, uma vez que, sua composição e concentração são conhecidas. Além disso, foi o primeiro relato do uso do SYBR Gold como corante fluorescente em análises de qPCR. A patente da mistura reacional desenvolvida se encontra depositada no Instituto Nacional de Propriedade Industrial (INPI), número de processo BR 10 2017 022164 4Abstract: Real-time PCR or quantitative PCR (qPCR) is widely used for DNA quantification and presents a high cost in biological research, mainly using of imported reagents. The objective was to develop a low-cost reaction mixture for Quantitative PCR (qPCR) with specificity, efficiency, and sensitivity equivalent to the imported commercial kits. Mixtures were assembled with varied concentrations of SYBR® Green or SYBR® Gold, MgCl2 and Taq DNA Polymerase Brazil (core reagents). The additives bovine serum albumin, betaine, dimethyl sulfoxide, formamide, polyethylene glycol 400, polyoxyethylene-octyl-phenyl-ether, trehalose and polysorbate-20 were added alone or combined and evaluated as reactions enhancers. During the developing process, reactions were conducted with genomic DNA (300 to 0.003 ng) of Porphyromonas gingivalis and specie-specific primers. To ensure applicability, the mixtures with best results were evaluated against DNA from other species: Streptococcus sanguinis, Mus Musculus and Candida albicans. Cycle threshold values and melting curves were used to compare mixtures to the commercial Power SYBR Green qPCR Master Mix Kit©. The best mixtures were tested for their stability. The mixture with better efficiency, specificity and stability was called non-commercial qPCR Master Mix. The efficiency of the non-commercial qPCR Master Mix was up to 28% higher than the commercial product, varying according to the concentration, origin and quality of the genetic material. The actual price of the qPCR can be reduced by up to 70% by employing the non-commercial qPCR Master Mix. In conclusion, a reaction mixture was developed for PCR, with high efficiency and specificity, and a low cost. The mix allows individual changes, since their composition and concentration are known. In addition, it was the first report of using SYBR Gold as a fluorescent dye in qPCR analyzes. The developed reaction mixture patent is in the National Institute of Industrial Property, process number BR 10 2017 022164 4DoutoradoMicrobiologia e ImunologiaDoutora em Biologia Buco-Dental154620/2014-92017/03263-3CNPQFAPES

Doctor of Philosophy

dissertationLittle is known about the kinetic limitations of the polymerase chain reaction (PCR). Advancements in chemistry and instrumentation have increased its speed and specificity. Further improvements will be facilitated by a more complete understanding of the rates of the individual reactions that comprise PCR. A continuous fluorescent assay is developed to study DNA polymerase extension. Nucleotide incorporation is monitored with noncovalent DNA dyes using a defined hairpin template. The extension rate is measured in nucleotides incorporated per second per molecule of polymerase and has greater relevance to PCR than traditional activity methods. This assay was developed and validated on a stopped-ow instrument and subsequently adapted for real-time PCR instruments to extend its utility to any laboratory setting. The influences of a variety of buer components were determined and optimal conditions for fast polymerase extension are recommended. The incorporation rates of each nucleotide were determined and extension was found to depend on template sequence. When DMSO was included in the reaction to reduce inhibition from secondary structure, extension rates of random sequences were closely approximated by their base composition. Extension rates as a function of temperature were determined and were applied to a kinetic model. This model accounts for extension during temperature transitions and more accurately portrays fast PCR with rapid thermal cycling. A complete model of PCR based on differential equations derived from mass action equations is provided. This can be used to incorporate experimentally derived parameters obtained for the other reactions of PCR. Knowledge of the temperature and chemistry dependence of reaction rates will enable improved thermal cycling and solution conditions for more rapid and effcient PCR

Exponential Megapriming PCR (EMP) Cloning-Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.National Institutes of Health (U.S.) (Grant GM077537

Towards the absolute quantification of DNA by PCR

Amplification techniques such as the Polymerase Chain Reaction (PCR) are held to be largely qualitative procedures and are widely used as such. Since the efficiency of amplification is less than perfect, small changes in efficiency can yield dramatic differences in the final amount of product generated. Despite this unpredictability the exquisite sensitivity of PCR makes the demanding goal of absolute quantification highly desirable. Consequently, the use of this technique for the quantification of nucleic acids has increased at an exponential rate. However, the ability of PCR to accurately quantify absolute levels of DNA is still not universally accepted. The overall aim of this investigation was to determine the critical factors affecting the quantification of DNA using PCR and to use these findings to develop an assay for the absolute quantification of DNA in a model system. The novel work presented here illustrates the need for careful examination of sequences for GC-rich domains which could give rise to stable secondary structures and reduce the efficiency of amplification by serving as termination sites. To determine the accuracy of competitive PCR, CE and IP-RP-HPLC were employed to quantify PCR- products. These two techniques provided valuable information on the identification and elimination of sources of error which led to improvements in speed, accuracy and precision, as well as ease of quantification by PCR. They also yielded information on the process of heteroduplex formation whilst simultaneously revealing assay limitations. Consequently, the on-line fluorescence monitoring of PCR was used as an alternative method for the quantification of Legionella pneumophila. This technique was highly reproducible however, mispriming and the subsequent amplification of non-specific PCR products limited the level of detection. The Y-end labelling of degraded DNA with DIG prevented short DNA fragments from mispriming (and consequently extending) allowing the amplification of DNA targets. Therefore, to reduce mispriming and hence improve assay sensitivity, this approach was adapted for the first time to produce 5'-degenerate, 3'- DIG-terminated competitive primer analogues. These analogues, coupled with the use of the LightcyclerTM, allowed the detection and absolute quantification of a single cell of Legionella pneumophila. This is the first time that this level of sensitivity has been achieved using this type of assay. This technique should provide a very rapid and sensitive alternative for quantification compared to the other, more expensive technologies available at present

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions