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.     

Using real-time PCR to determine transgene copy number in wheat

Transgene copy number is usually determined by means of Southern blot analysis which can be time consuming and laborious. In this study, quantitative real-time PCR was developed to determine transgene copy number in transgenic wheat. A conserved wheat housekeeping gene,puroindoline-b, was used as an internal control to calculate transgene copy number. Estimated copy number in transgenic lines using real-time quantitative PCR was correlated with actual copy number based on Southern blot analysis. Real-time PCR can analyze hundreds of samples in a day, making it an efficient method for estimating copy number in transgenic wheat.     

High-throughput transgene copy number estimation by competitive PCR

Transgene copy number affects the level and stability of gene expression. Therefore, it is important to determine the copy number of each transgenic line. Polymerase chain reaction (PCR) is widely employed to quantify amounts of target sequences. Although PCR is not inherently quantitative, various means of overcoming this limitation have been devised. Recent real-time PCR methods are rapid; however, they typically lack a suitable internal standard, limit the size of the target sequence, and require expensive specialized equipment. Competitive PCR techniques avoid these problems, but traditional competitive methods are time consuming. Here we apply mathematical modeling to create a rapid, simple, and inexpensive copy number determination method that retains the robustness of competitive PCR.     

质粒拷贝数的测定方法

在研究生物工程重组蛋白产量的过程中 ,质粒的拷贝数是一个需要重点考虑的参数 ,对它进行精确定量至关重要。本文概述了几种主要的测定方法的原理和特点。     

Probe-free allele-specific copy number detection and analysis of tumors

Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping.     

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte‐colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra‐assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild‐type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin‐based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue‐specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1390–1400, 2014     

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.     

番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用

以番茄(Solanum lycopersicum L.cv.Micro-Tom)叶片为试材,建立了一种简便快速制备叶片基因组DNA的方法。2~20 mm2的叶片即可满足制备要求,制备过程只需一种提取试剂、只涉及1次移液和1次离心操作,不涉及沉淀。确定了所制备的DNA用于实时荧光定量PCR的合适用量为0.1~0.2μL(反应总体积为12.5μL),发现过量模板的使用可降低PCR效率且可导致扩增失败。该项DNA快速制备及相适应的实时荧光定量PCR技术已成功应用于番茄转基因植株检测。     

实时荧光定量PCR(TaqMan)法测定外源基因的拷贝数

实时荧光定量PCR是近年新兴的一项技术,因其快速、方便、便宜,需要DNA样品量少,无需放射性检测等优点被广泛应用于基因的定量分析。该文就实时荧光定量PCR(TaqMan)技术的发展、基本原理及测定外源基因拷贝数的技术流程做一介绍。     

Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assay's discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.     

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.     

Variable copy number of macronuclear DNA molecules in Tetrahymena

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.     

tRNA gene copy number variation in humans

The human tRNAome consists of more than 500 interspersed tRNA genes comprising 51 anticodon families of largely unequal copy number. We examined tRNA gene copy number variation (tgCNV) in six individuals; two kindreds of two parents and a child, using high coverage whole genome sequence data. Such differences may be important because translation of some mRNAs is sensitive to the relative amounts of tRNAs and because tRNA competition determines translational efficiency vs. fidelity and production of native vs. misfolded proteins. We identified several tRNA gene clusters with CNV, which in some cases were part of larger iterations. In addition there was an isolated tRNALysCUU gene that was absent as a homozygous deletion in one of the parents. When assessed by semiquantitative PCR in 98 DNA samples representing a wide variety of ethnicities, this allele was found deleted in hetero- or homozygosity in all groups at ~ 50% frequency. This is the first report of copy number variation of human tRNA genes. We conclude that tgCNV exists at significant levels among individual humans and discuss the results in terms of genetic diversity and prior genome wide association studies (GWAS) that suggest the importance of the ratio of tRNALys isoacceptors in Type-2 diabetes.     

An efficient and reliable method for determining the number of transgenes inserted into transgenic plants

A simple and reliable method is described for determining the number of transgenes inserted into transgenic plants. Analysis of tomato plants transformed with an antisense polygalacturonase gene is used to demonstrate the method. The use of the method to analyze other plants transformed with endogenous plant gene coding regions or promoters is discussed.     

Detection of Stachybotrys chartarum using rRNA, tri5, and β-tubulin primers and determining their relative copy number by real-time PCR

Highly conserved regions are attractive targets for detection and quantitation by PCR, but designing species-specific primer sets can be difficult. Ultimately, almost all primer sets are designed based upon literature searches in public domain databases, such as the National Center for Biotechnology Information (NCBI). Prudence suggests that the researcher needs to evaluate as many sequences as available for designing species-specific PCR primers. In this report, we aligned 11, 9, and 16 DNA sequences entered for Stachybotrys spp. rRNA, tri5, and β-tubulin regions, respectively. Although we were able to align and determine consensus primer sets for the 9 tri5 and the 16 β-tubulin sequences, there was no consensus sequence that could be derived from alignment of the 11 rRNA sequences. However, by judicious clustering of the sequences that aligned well, we were able to design three sets of primers for the rRNA region of S. chartarum. The two primer sets for tri5 and β-tubulin produced satisfactory PCR results for all four strains of S. chartarum used in this study whereas only one rRNA primer set of three produced similar satisfactory results. Ultimately, we were able to show that rRNA copy number is approximately 2-log greater than for tri5 and β-tubulin in the four strains of S. chartarum tested.     

Number matters: control of mammalian mitochondrial DNA copy number

Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.     

Transgene copy number distribution profiles in recombinant CHO cell lines revealed by single cell analyses

Clonally derived recombinant cell lines are highly desired to achieve consistent production of recombinant biotherapeutics. Despite repeated rounds of cloning by limiting dilution or single cell cloning, the resulting cell lines have often been observed to diverge, becoming a heterogeneous population and losing productivity over long-term sub-culturing. To understand the underlying molecular mechanisms, we developed quantitative polymerase chain reaction (qPCR) assays for the analysis of transgene copy number distribution in single recombinant cells isolated from Chinese hamster ovary (CHO) cell lines. Single cells were obtained by fluorescence activated cell sorting (FACS) technology and lysed directly in 96-well plates. qPCR assays were then applied to analyze the quantity and distribution of transgenes in those single cells. Results revealed multiple types of transgene copy number distribution profiles from those clonally derived CHO cell lines. The cell lines that maintained productivity over time displayed relatively constant and homogeneous transgene copy number distributions; while most of those cell lines exhibiting a loss of productivity over time showed varying degrees of transgene copy number heterogeneity and distribution drift with passaging. Some cell lines showed the existence of a significant portion of cells lacking the transgenes (referred to as negative cells in this study) and the percentage of those negative cells increased with subsequent generations. Criteria based on transgene copy number distribution profiles were developed to assess cell line suitability for clinical applications, which include (i) percentage of negative cells; (ii) standard deviation of qPCR threshold cycle (C(t) ) value, a measure of population heterogeneity; (iii) mean C(t) changes during aging, a measure of population drift. By implementing these criteria, undesirable cell lines were eliminated for further clinical and commercial applications.