Estimation of tumor heterogeneity using CGH array data

Background  

Array-based comparative genomic hybridization (CGH) is a commonly-used approach to detect DNA copy number variation in whole genome-wide screens. Several statistical methods have been proposed to define genomic segments with different copy numbers in cancer tumors. However, most tumors are heterogeneous and show variation in DNA copy numbers across tumor cells. The challenge is to reveal the copy number profiles of the subpopulations in a tumor and to estimate the percentage of each subpopulation.     

PanGEA: Identification of allele specific gene expression using the 454 technology

Background  

Next generation sequencing technologies hold great potential for many biological questions. While mainly used for genomic sequencing, they are also very promising for gene expression profiling. Sequencing of cDNA does not only provide an estimate of the absolute expression level, it can also be used for the identification of allele specific gene expression.     

The Average Mutual Information Profile as a Genomic Signature

Background  

Occult organizational structures in DNA sequences may hold the key to understanding functional and evolutionary aspects of the DNA molecule. Such structures can also provide the means for identifying and discriminating organisms using genomic data. Species specific genomic signatures are useful in a variety of contexts such as evolutionary analysis, assembly and classification of genomic sequences from large uncultivated microbial communities and a rapid identification system in health hazard situations.     

Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase

Background

Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.

Methodology/Principal Findings

We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.

Conclusions/Significance

The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.     

Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard: Microalgal pcna as the Model Gene

Quantitative real-time PCR (qPCR) has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear) used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form) and linear DNA standards (linearized plasmid DNA or PCR amplicons), using proliferating cell nuclear gene (pcna), the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.     

Femtosecond laser treatment enhances DNA transfection efficiency in vivo

Background  

Gene therapy with plasmid DNA is emerging as a promising strategy for the treatment of many diseases. One of the major obstacles to such therapy is the poor transfection efficiency of DNA in vivo.     

A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR

Background

Based upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach.

Results

Recognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of ± 25% can be routinely achieved. Comparison with the LinReg and Miner automated qPCR data processing packages further demonstrated the superior performance of this kinetic-based methodology.

Conclusion

Called "linear regression of efficiency" or LRE, this novel kinetic approach confers the ability to conduct high-capacity absolute quantification with unprecedented quality control capabilities. The computational simplicity and recursive nature of LRE quantification also makes it amenable to software implementation, as demonstrated by a prototypic Java program that automates data analysis. This in turn introduces the prospect of conducting absolute quantification with little additional effort beyond that required for the preparation of the amplification reactions.     

Limited genetic diversity in <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Enteritidis PT13

Background  

Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.     

Non-viral eNOS gene delivery and transfection with stents for the treatment of restenosis

Background  

In this study, we have examined local non-viral gene delivery, transfection, and therapeutic efficacy of endothelial nitric oxide synthase (eNOS) encoding plasmid DNA administered using coated stents in a rabbit iliac artery restenosis model.     

Sequence periodicity of Escherichia coli is concentrated in intergenic regions

Background  

Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property. This genomic feature was demonstrated for many prokaryotic genomes. The Escherichia coli sequences display the period close to 11 base pairs.     

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

Background  

Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci.     

Cpf1-assisted efficient genomic integration of in vivo assembled DNA parts in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objectives

To test the applicability of Cpf1 from Francisella novivida in genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.

Results

An easy-to-use vector toolkit, containing a CEN6/ARS4 plasmid expressing Cpf1 from Francisella novivida (FnCpf1) and a 2 μ plasmid for crRNA or crRNA array expressing, was constructed for Cpf1-assisted genomic integration in S. cerevisiae. Our results showed that FnCpf1 allowed for targeted singleplex, doubleplex, and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 95, 52, and 43%, respectively.

Conclusions

CRISPR-Cpf1 system allows for efficient genomic integration of in vivo assembled DNA parts in S. cerevisiae, and thus provides an alternative CRISPR-Cas method for metabolic pathway engineering in addition to CRISPR-Cas9 system previously reported for yeast.

     

High-throughput isolation of ultra-pure plasmid DNA by a robotic system

Background  

With the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA.     

Pellicle associated adherence film above incubation broth surface - an inexpensive adjunct to recognizing Candida krusei in the laboratory

Background

Targeting Induced Local Lesions in Genomes (TILLING) is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels) in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate.

Findings

A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C.

Conclusions

A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.     

A fixed-point algorithm for estimating amplification efficiency from a polymerase chain reaction dilution series

Background

The polymerase chain reaction amplifies and quantifies small amounts of DNA. It is a cyclic process, during each cycle of which each strand of template DNA is copied with probability approaching one: the amount of DNA approximately doubles and this amount can be estimated fluorimetrically each cycle, producing a set of fluorescence values hereafter referred to as the amplification curve. Commonly the biological question of relevance is one of the ratio of DNA concentrations in two samples: a ratio that is deduced by comparing the two amplification curves, usually by way of a plot of fluorescence against cycle number. Central to this analysis is measuring the extent to which one amplification curve is shifted relative to the other, a measurement often accomplished by defining a threshold or quantification cycle, C_q, for each curve: the fractional cycle number at which fluorescence reaches some threshold or at which some other criterion (maximum slope, maximum rate of change of slope) is satisfied.We propose an alternative where position is measured relative to a reference curve; position equates to the cycle shift which maximizes the correlation between the reference and the observed fluorescence sequence. A key parameter of the reference curve is obtained by fixed-point convergence.

Results

We consider the analysis of dilution series constructed for the estimation of qPCR amplification efficiency. The estimate of amplification efficiency is based on the slope of the regression line when the C_q is plotted against the logarithm of dilution. We compare the approach to three commonly used methods for determining C_q; each is applied to publicly accessible calibration data sets, and to ten from our own laboratory. As in the established literature we judge their relative merits both from the standard deviation of the slope of the calibration curve, and from the variance in C_q for replicate fluorescence curves.

Conclusions

The approach does not require modification of experimental protocols, and can be applied retrospectively to existing data. We recommend that it be added to the methodological toolkit with which laboratories interpret their real-time PCR data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0372-4) contains supplementary material, which is available to authorized users.     

A calibration method for patient specific IMRT QA using a single therapy verification film

Aim

The aim of the present study is to develop and verify the single film calibration procedure used in intensity-modulated radiation therapy (IMRT) quality assurance.

Background

Radiographic films have been regularly used in routine commissioning of treatment modalities and verification of treatment planning system (TPS). The radiation dosimetery based on radiographic films has ability to give absolute two-dimension dose distribution and prefer for the IMRT quality assurance. However, the single therapy verification film gives a quick and significant reliable method for IMRT verification.

Materials and methods

A single extended dose rate (EDR 2) film was used to generate the sensitometric curve of film optical density and radiation dose. EDR 2 film was exposed with nine 6 cm × 6 cm fields of 6 MV photon beam obtained from a medical linear accelerator at 5-cm depth in solid water phantom. The nine regions of single film were exposed with radiation doses raging from 10 to 362 cGy. The actual dose measurements inside the field regions were performed using 0.6 cm³ ionization chamber. The exposed film was processed after irradiation using a VIDAR film scanner and the value of optical density was noted for each region. Ten IMRT plans of head and neck carcinoma were used for verification using a dynamic IMRT technique, and evaluated using the gamma index method with TPS calculated dose distribution.

Results

Sensitometric curve has been generated using a single film exposed at nine field region to check quantitative dose verifications of IMRT treatments. The radiation scattered factor was observed to decrease exponentially with the increase in the distance from the centre of each field region. The IMRT plans based on calibration curve were verified using the gamma index method and found to be within acceptable criteria.

Conclusion

The single film method proved to be superior to the traditional calibration method and produce fast daily film calibration for highly accurate IMRT verification.     

Routes to improving the reliability of low level DNA analysis using real-time PCR

Background  

Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated.