M.xanthus Dk1622 Genome Sequence Analysis And New Random Degenerate Primers Database Build

Two main parts are introduced in this thesis. The first part is mainly about the analysis of microbial genome sequence, and the second part is about the innovative idea on how to design AD primers, as well as the construction of AD primer database for Microbes (ADDB).The analysis of microbial genome sequences depend on the means of bioinformatics which can be divided into two parts: data administration and data analysis.Data administration refers to the administration of molecular data such as nucleic acids sequences, protein sequences and so on by means of database methods. Several databases which include a serial of nucleic acids sequences databases, protein sequences databases, 3D structure of large biological molecules databases, protein classification databases, genome databases and so on are introduced in brief in this thesis.Data analysis is a process of mining information from a great deal of data by constructing mathematic model and programming. Data analysis of microbial genomes contains single genome analysis and genomic comparison. Single genome analysis contains genomic sequence annotation which mainly refers to finding ORFs, rRNA and tRNA, and the prediction of genes' functions and function classification, and analysis of genomic components which refers to base composition analysis, codon usage bias and so on. Comparative genome analysis not only contains the comparison of basic characters of genome but also contains phylogenetic analysis.The complete sequence of Myxococcus xanthus DK.1622 which did not contain annotation is released by TIGR in November, 2003. Annotation of DK1622 and relative analysis of Myxococcus xanthus DK1622 sequence were carried on to get predominance in the research of important genes.The innovative idea of AD primer design is on the foundation of statistics of oligonucleotides' appearance frequency in microbial genomes.Thermal Asymmetric Interlaced PCR (TA1L-PCR) serves as a simple, effective and widely applied method to isolate DNA segments adjacent to known sequences. However, there is no standard principle of designing the arbitrary degenerated (AD) primers which play a very important role in the process of TAIL-PCR. A set of AD primers, no more than twenty, was used repeatedly in published papers. Considering the different nucleotide composition among species, existing AD primers are prone to be ineffective, which greatly constrain the application of TAIL-PCR. To solve this problem, AD Primer Database for Microbe (ADDB) was constructed and until now, 301 sets of AD primers were generated according to 301 sequenced microbial genomes, and every set of AD primers can apply to species homologous to the model genome or those having similar GC contents. Moreover, the AD primers which were designed according to the sequenced genome Myxococcus xanthus DK1622 in ADDB have be proved more effective than reported primers in Myxococcus flu. HW-1, Sorangium and Streptomyces in laboratory validation.