Chromatography of plasmid DNA

Liquid chromatography plays a central role in process-scale manufacturing of therapeutic plasmid DNA (pDNA) for gene therapy and DNA vaccination. Apart from its use as a preparative purification step, it is also very useful as an analytical tool to monitor and control pDNA quality during processing and in final formulations. This paper gives an overview of the use of pDNA chromatography. The specificity of pDNA purification and the consequent limitations to the performance of chromatography are described. Strategies currently used to overcome those limitations, as well as other possible solutions are presented. Applications of the different types of chromatography to the purification of therapeutic pDNA are reviewed, and the main advantages and disadvantages behind each technique highlighted.     

Direct selective detection of genomic DNA from coliform using a fiber optic biosensor

Fiber optic biosensors operated in a total internal reflection format were prepared based on covalent immobilization of 25mer lacZ single-stranded nucleic acid probe. Genomic DNA from Escherichia coli was extracted and then sheared by sonication to prepare fragments of approximately 300mer length. Other targets included a 25mer fully complementary lacZ sequence, 100mer polymerase chain reaction (PCR) products containing the lacZ sequence at various locations, and non-complementary DNA including genomic samples from salmon sperm. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on extent or speed of hybridization of complementary oligonucleotides. Detection of genomic fragments containing the lacZ sequence was possible in as little as 20 s by observation of the steady-state fluorescence intensity increase or by time-dependent rate of fluorescence intensity changes.     

Analysis of plant genomic DNAs and the genetic relationship among plants by using surface-enhanced Raman spectroscopy

The study aimed to distinguish genomic DNAs from nine species of plants belonging to six families and analyze their genetic relationship by using surface-enhanced Raman scattering (SERS). The silver nano-colloid and excitation wavelength of 785 nm used in this study yielded excellent quality of the SERS spectra. Raman signals were remarkably enhanced. Although the spectra for the nine species of plants appeared very similar, there were significant differences according to the analysis of variance analysis. There were three strong characteristic peaks. The peak at 625 cm⁻¹ was due to the vibration overlap of C3′-endo/anti deoxyribose, cytosine, and guanine; the one at 715 cm⁻¹ was due to the scissoring vibrations of C2N1C6 of adenine; and that at 1011 cm⁻¹ was due to the stretching vibration of the CO bond of deoxyribose and vibrations of cytosine. The SERS data were smoothed and standardized and evaluated using second derivative analysis, principal component analysis, and hierarchical cluster analysis. A model was established using the data from hierarchical cluster analysis and principal components of the second derivative. The clustering result of this model was highly consistent with the traditional classification of plants; all plant species investigated were correctly clustered into classes according to the cluster distance coefficient among them; the accuracy of clustering was 100%. Chinese cabbage (Brassica pekinensis Rupr.) and green cabbage (Brassica chinensis L.) belonging to Cruciferae, maize (Zea mays L.) and bamboo (Sinocalamus affinis McClure) belonging to Gramineae, and magnolia (Magnolia delavayi Franch.) and champaca (Michelia alba DC.) belonging to Magnoliaceae were clustered into three separate classes, and fern (Nephrolepis auriculata L., Nephrolepidaceae), garlic (Allium sativum L., Amaryllidaceae), and ginkgo (Ginkgo biloba L., Ginkgoaceae) were each clustered into separate classes. These findings suggest that the SERS spectra of plant genomic DNAs can be used to classify species and analyze their genetic relationship. It is an effective and perfect supplement to traditional classification and can form the basis for genetic analysis.     

Differentially expressed genes selection via Laplacian regularized low-rank representation method

With the rapid development of DNA microarray technology and next-generation technology, a large number of genomic data were generated. So how to extract more differentially expressed genes from genomic data has become a matter of urgency. Because Low-Rank Representation (LRR) has the high performance in studying low-dimensional subspace structures, it has attracted a chunk of attention in recent years. However, it does not take into consideration the intrinsic geometric structures in data.In this paper, a new method named Laplacian regularized Low-Rank Representation (LLRR) has been proposed and applied on genomic data, which introduces graph regularization into LRR. By taking full advantages of the graph regularization, LLRR method can capture the intrinsic non-linear geometric information among the data. The LLRR method can decomposes the observation matrix of genomic data into a low rank matrix and a sparse matrix through solving an optimization problem. Because the significant genes can be considered as sparse signals, the differentially expressed genes are viewed as the sparse perturbation signals. Therefore, the differentially expressed genes can be selected according to the sparse matrix. Finally, we use the GO tool to analyze the selected genes and compare the P-values with other methods.The results on the simulation data and two real genomic data illustrate that this method outperforms some other methods: in differentially expressed gene selection.     

Interorganellar DNA transfer in wheat: dynamics and phylogenetic origin

A homology search of wheat chloroplast (ct) and mitochondrial (mt) genomes identified 54 ctDNA segments that have homology with 66 mtDNA segments. The mtDNA segments were classified according to their origin: orthologs (prokaryotic origin), xenologs (interorganellar DNA transfer origin) and paralogs (intraorganellar DNA amplification origin). The 66 mtDNA sequences with homology to ctDNA segments included 14 paralogs, 18 orthologs and 34 xenologs. Analysis of the xenologs indicated that the DNA transfer occurred unidirectionally from the ct genome to the mt genome. The evolutionary timing of each interorganellar DNA transfer that generated a xenolog was estimated. This analysis showed that 2 xenologs originated early in green plant evolution, 4 in angiosperm evolution, 3 in monocotyledon evolution, 9 during cereal diversification and 8 in the evolution of wheat. Six other xenologs showed recurrent transfer from the ct to mt genomes in more than one taxon. The two remaining xenologs were uninformative on the evolutionary timing of their transfer. The wheat mt nad9 gene was found to be chimeric, consisting of the cereal nad9 gene and its 291 bp 5'-flanking region that included a 58 bp xenolog of the ct-ndhC origin.     

Up a gear? The significance of an elevated mutation rate in tumorigenesis

Mutations involved in many cancers have been identified, but with some cancers requiring six or more mutations to take on their fully metastatic forms, the question remains whether all of these mutations can be acquired via a process of successive mutation, at a normal rate, and clonal expansion or whether heightened mutation rates are required. This issue has been debated for decades. Recently there has been much interest in forms of genomic instability such as chromosomal instability and microsatellite instability. It remains to definitively show whether or not these instabilities are very early causal events in tumorigenesis. This article reviews the evidence for and against genomic instability being an early causal event in tumorigenesis and surveys the mathematical modelling literature in this area. The focus is on chromosomal instability and microsatellite instability in colorectal cancer.     

Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.     

三种烟草品种基因组DNA傅里叶红外光谱研究

利用傅里叶红外光谱(FTIR)研究三种烟草品种DNA的差异,旨在对烟草品种进行亲缘关系分析和品种鉴定。研究结果显示,三种烟草品种DNA红外光谱较为相似,均有四个明显的特征峰,1 103 cm~(-1)是DNA磷酸二酯键的对称伸缩振动,1 236 cm~(-1)是DNA磷酸二酯键的非对称伸缩振动,1 400 cm~(-1)是DNA糖苷键的伸缩振动,I 622 cm~(-1)是DNA中是胞嘧啶C4—C5=C6的环伸缩振动。通过平滑、标准化处理、二阶求导、提取主成分和系统聚类分析建立了DNA FTIR光谱数据聚类分析模型。使用该模型对三个烟草品种进行鉴定,鉴定正确率为100%。使用该模型对三个烟草品种进行亲缘关系分析,云烟87与K326聚为一类,距离系数为0.003,DNA相似度为99.7%,红大单独聚为一类。聚类正确率达100%。该项研究为烟草品种鉴定及遗传育种提供了参考。     

基因组重组问题的一个更快算法

寻找一个基因组(源基因组)转化成另一个基因组(目标基因组)所需最少数目移位和翻转的问题,称为基因组重组问题.此问题的“瓶颈”在于寻找源基因组的一个最优“联接”;若源基因组和目标基因组是“共尾”的,Hannenhalli和Pevzner给出一个O(n2)算法得到源基因组的一个最优“联接”,本文将此算法复杂性将低到O(n),其中n为基因组中所含基因的个数.从而由Eric.T和MarieFrance的结果得到求“共尾”标号基因组间重组序列的一个O(nnlogn)算法.