Rapid PCR-based determination of transgene copy number in rice

We present a simple, rapid, and low-cost method to determine transgene copy number in rice. More than 100 first- and second-generation transgenic rice plants were tested. The plasmid (pRCopy) used for rice transformation contains the specific gene of interest and a partially deleted cytochrome c gene (cyc), a single-copy gene in rice. A 132-bp segment of the cloned ricecyc was shortened to 108 bp by deleting a 24-bp internal fragment. After PCR amplification of the genomic DNA from transgenic rice harboring pRCopy, the 2 expected bands were found. The 121-bp band corresponds to the endogenouscyc; the 97-bp band comes from the integrated pRCopy. Clear distinctions can be made between single and multiple copies of the transgene by comparing band densities.     

Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria

A method for determination of plasmid copy number (PCN) in bacteria by real-time quantitative polymerase chain reaction (QPCR) was developed as an alternative to current PCN assays. Conventional methods for PCN estimation are generally not of high throughput, laborious, have low reproducibility, require large amounts of biological samples and are applicable only for a narrow dynamic range. Real-time QPCR, using the ABI Prism 7000, was able to sensitively detect the quantity of the pUC ori based plasmid, NS3, transformed into Escherichia coli host, DH5alpha, to be 411+/-6.1. The PCN of pBR322 plasmid DNA in DH5alpha was estimated to be 40+/-0.6 which is within its previously reported PCN range of approximately 30 to 70. QPCR was found to show good reproducibility and high sensitivity in detecting a two fold difference in template concentration, and a wide linear dynamic range covering 0.5 pg to 50 ng of DNA. PCNs of DH5alpha bearing plasmids pBR322 and NS3 computed from real-time QPCR assay were validated by that of agarose gel assay, and a marginal difference of only 13.0% and 10.7% was found for the two plasmids respectively. The QPCR assay was able to detect changes in PCN of plasmid producing DH5alpha during the course of a 2 l batch fermentation.     

Plasmid copy number and stability determination in Clostridium perfringens transformants

The copy number of the streptococcal plasmid pAM beta 1 (26.5 kb), and its deletion derivatives, pVA1 (11 kb) and pVA677 (7.6 kb) contained in Clostridium perfringens 3624A transformants was determined by incorporation of [methyl-3H]thymidine (4 muCi/ml) into chromosomal and plasmid DNA and sizing of the C. perfringens genome using transverse alternating field electrophoresis. Plasmids pAM beta 1, pVA1, and pVA677 were found to be present at 1.0, 97, and 216 copies/cell, respectively. 10.2, 54, and 96% of the initial pAM beta 1-, pVA1- and pVA677-containing transformants, respectively, remained resistant to erythromycin over 220 generations of growth. The results indicate a size-dependent relationship between plasmid stability and plasmid copy number in C. perfringens.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

Simple assay for quantitation of Tn5 inversion events in Escherichia coli and use of the assay in determination of plasmid copy number.

In this report, we describe a simple method for measuring the frequency of sequence inversion in the transposable element Tn5 as a result of recombination across its duplicated IS50 elements. The structure of Tn5 was manipulated so that the neomycin phosphotransferase gene of the transposon would be expressed only if a sequence inversion event occurred. This highly sensitive assay also served as the basis for a novel means of estimating plasmid copy number.     

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

Background  

Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed.     

Solid-phase minisequencing confirmed by FISH analysis in determination of gene copy number

The solid-phase minisequencing method (Syvänen et al. 1990) allows accurate quantative determination of the ratio between two DNA or RNA sequences that are present as a mixture in a sample and differ from each other only by a single nucleotide. Here, we present another application of the minisequening method, the determination of the gene copy number in a genome. The copy number of a marker gene aspartyl glucosaminidase (AGA) located at 4qter, was determined in three patients with a chromosomal alteration involving the distal region of 4q. For the minisequencing assay an equal amount of DNA from a patient homozygous for a mutation in the AGA gene was added to the DNA samples concerned. The relative amount of the normal sequence determined in each combined sample gives the copy number of the AGA gene. Fluorescence in situ hybridization (FISH), applied in parallel as a control, produced concordant results with solid-phase minisequencing in each case. As the potential of the minisequencing lies in automation, it could be a useful tool in the screening of monosomies, trisomies or loss of heterozygosity in diagnostics.     

Analysis of gene copy number changes in tumor phylogenetics

Backgound

Evolution of cancer cells is characterized by large scale and rapid changes in the chromosomal  landscape. The fluorescence in situ hybridization (FISH) technique provides a way to measure the copy numbers of preselected genes in a group of cells and has been found to be a reliable source of data to model the evolution of tumor cells. Chowdhury et al. (Bioinformatics 29(13):189–98, 23; PLoS Comput Biol 10(7):1003740, 24) recently develop a computational model for tumor progression driven by gains and losses in cell count patterns obtained by FISH probes. Their model aims to find the rectilinear Steiner minimum tree (RSMT) (Chowdhury et al. in Bioinformatics 29(13):189–98, 23) and the duplication Steiner minimum tree (DSMT) (Chowdhury et al. in PLoS Comput Biol 10(7):1003740, 24) that describe the progression of FISH cell count patterns over its branches in a parsimonious manner. Both the RSMT and DSMT problems are NP-hard and heuristics are required to solve the problems efficiently.

Methods

In this paper we propose two approaches to solve the RSMT problem, one inspired by iterative methods to address the “small phylogeny” problem (Sankoff et al. in J Mol Evol 7(2):133–49, 27; Blanchette et al. in Genome Inform 8:25–34, 28), and the other based on maximum parsimony phylogeny inference. We further show how to extend these heuristics to obtain solutions to the DSMT problem, that models large scale duplication events.

Results

Experimental results from both simulated and real tumor data show that our methods outperform previous heuristics (Chowdhury et al. in Bioinformatics 29(13):189–98, 23; Chowdhury et al. in PLoS Comput Biol 10(7):1003740, 24) in obtaining solutions to both RSMT and DSMT problems.

Conclusion

The methods introduced here are able to provide more parsimony phylogenies compared to earlier ones which are consider better choices.

     

Analysis of copy number variants in the cattle genome

Copy number variation (CNV) is likely to be an important component of heritable variation in livestock. To characterise CNVs in cattle, we performed a genome wide survey to determine the number, location and gene content of these genomic features. A tiling oligonucleotide array with ~385,000 probes was used for comparative genomic hybridisation of both taurine and zebu cattle. Using a conservative set of calling criteria, a total of 51 CNV were detected that collectively spanned approximately half of one percent of the bovine genome. The size of the average CNV within each animal ranged from 213 kb up to 335 kb. Half of the CNV were detected in a single animal only, whilst the remainder was independently identified in multiple individuals. Analysis was performed to determine the gene content for each CNV region. This revealed that the majority of CNV (82%) spanned at least one gene, with a number of CNV containing genes which are known to control aspects of phenotypic variation in cattle. Whilst additional studies are required to determine the impact of individual CNV, this study confirmed them as an important class of genomic variation in cattle.     

Detection of differential gene copy number using denaturing high performance liquid chromatography

Genomic rearrangements leading to deletion or duplication of gene(s) resulting in alterations in gene copy number underlie the molecular lesion in several genetic disorders. Methods currently used to determine gene copy number including real time PCR, southern hybridization, fluorescence in situ hybridization, densitometric scanning of PCR product etc. have certain disadvantages and are also expensive and time consuming. Herein, we describe a simple and rapid method to assess gene copy number using denaturing high performance liquid chromatography (dHPLC). We used X chromosome genes as model to compare the gene copy numbers present on this chromosome in males and females. DNA from these samples were amplified by biplex PCR using primer pairs specific for X chromosome genes only (target gene) and for genes present on both X and Y chromosomes (internal control). Amplified products were analyzed using HPLC under non-denaturing conditions. The ratio of peak areas (target gene/internal control) of the amplified products was approximately twice in female samples than male samples (p < 0.001) demonstrating that the differential gene copy number can be easily detected using this method. This method can potentially be used for diagnostic purpose where the need is to distinguish samples based on the differential gene copy numbers.     

Some stochastic models for plasmid copy number

Some stochastic models for the copy number of plasmids in a cell line are studied. When considering the behavior of copy number in the whole cell line, the theory of multitype branching processes is appropriate. Attention is paid to the cure rate in the cell line, and the asymptotic fractions of cells containing a given number of plasmids. These quantities are used to compare the models numerically.     

Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR

We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10⁴ cells are required for the analysis.     

Optimization of a quantitative PCR based method for plasmid copy number determination in human cell lines

Transient gene expression (TGE) is an essential tool for the production of recombinant proteins, especially in early drug discovery and development phases of biopharmaceuticals. The need for fast production of sufficient recombinant protein for initial tests has dramatically increased with increase in the identification of potential novel pharmaceutical targets. One of the critical factors for transient transfection is plasmid copy number (PCN), for which we here provide an optimized qPCR based protocol. Thereby, we show the loss of PCN during a typical batch process of HEK293 cells after transfection from 606,000 to 4560 copies per cell within 5 days. Finally two novel human kidney cell lines, RS and RPTEC/TERT1 were compared to HEK293 and proved competitive in terms of PCN and specific productivity.In conclusion, since trafficking and degradation of plasmid DNA is not fully understood yet, improved methods for analysis of PCN may contribute to design specific and more stable plasmids for high yield transient gene expression systems.     

Analysis of copy number variation using quantitative interspecies competitive PCR

Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.     

Absolute SILAC-compatible expression strain allows Sumo-2 copy number determination in clinical samples

Quantitative mass spectrometry-based proteomics is a vital tool in modern life science research. In contrast to the popularity of approaches for relative protein quantitation, the widespread use of absolute quantitation has been hampered by inefficient and expensive production of labeled protein standards. To optimize production of isotopically labeled standards, we genetically modified a commonly employed protein expression Escherichia coli strain, BL21 (DE3), to construct an auxotroph for arginine and lysine. This bacterial strain allows low-cost, high-level expression of fully labeled proteins with no conversion of labeled arginine to proline. In combination with a fluorescence-based quantitation of standards and nontargeted LC-MS/MS analysis of unfractionated total cell lysates, this strain was used to determine the copy number of a post-translational modifier, small ubiquitin-like modifier (SUMO-2), in HeLa, human sperm, and chronic lymphocytic leukemia cells. By streamlining and improving the generation of labeled standards, this production system increases the breadth of absolute quantitation by mass spectrometry and will facilitate a far wider uptake of this important technique than previously possible.     

Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age

Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy β-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 ± 920, significantly higher than that in skeletal muscle, 3650 ± 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.     

Molecular methods for genotyping complex copy number polymorphisms

Genome structural variation shows remarkable complexity with respect to copy number, sequence content and distribution. While the discovery of copy number polymorphisms (CNP) has increased exponentially in recent years, the transition from discovery to genotyping has proved challenging, particularly for CNPs embedded in complex regions of the genome. CNPs that are collectively common in the population and possess a dynamic range of copy numbers have proved the most difficult to genotype in association studies. This is in some part due to technical limitations of genotyping assays and the sequence properties of the genomic region being analyzed. Here we describe in detail the basis of a number of molecular techniques used to genotype complex CNPs, compare and contrast these approaches for determination of multi-allelic copy number, and discuss the potential application of these techniques in genetic studies.