A comparison of the standard DNA shotgun sequencing approach and the exponential DNA amplification method for sequencing BAC, fosmid, plasmid, and mitochondrial DNA

It has been almost three decades since the initial reports of Sanger and Coulson (Sanger, Nicklen, and Coulson, 1977) and Maxam and Gilbert (1977) described the enzymatic and chemical DNA sequencing methods, respectively. During this time, refinements of these methods have resulted in an increase of several orders of magnitude in the rate of DNA sequencing. These improvements have come about because individual DNA sequencing groups have made a conscious effort to not become satisfied with the technology, but have continuously worked to improve the methodology, while increasing accuracy and reducing the cost. The rational behind this philosophy is the target for DNA sequencing studies that have rapidly progressed from a few kilobase plasmid or cDNA to a multimegabase eukaryote genome. The increasing promise of gaining significant information directly related to the genetic basis of many living systems, as well as the role of the genome in normal and abnormal cellular development, has been the catalyst.;Therefore, during the initial phase of my studies, I became well versed in the standard shotgun sequencing techniques and completed the sequence of six regions of the human and mouse genome totaling over 1 Mbp. Then, because of my interest in forensics and the wide spread use of mitochondrial analysis in this field, I sequenced the entire ∼18 Kbp Zebrafish mitochondrial genome. However, it soon became apparent that one of the major troublesome areas in genomic studies is the ability to obtain highly purified DNAs that could serve as templates for further DNA sequencing. To investigate the idea of developing improved methods to obtain the desired, highly purified DNA needed to move the field forward, I therefore, investigated the possibility of amplifying DNA via the rolling circle amplification (Fire and Xu, 1995; Liu et al. 1996; Lizardi et al. 1998). My initial studies employed the TempliPhi (Dean et al. 2001) whole genome amplification procedure that was developed for amplifying plasmid-based clones directly from lysed bacterial cells. Although this TempliPhi protocol was used successfully for plasmid and BAC amplification, it lacked the specificity needed to prevent unwanted host genomic DNA amplification along with the plasmid or BAC clone that had been transformed into the bacterial host. Since the Phi-29 enzyme remained active at 0°C numerous artifacts were observed. Therefore, in an effort to overcome these limitations, I investigated replacing the Phi-29 enzyme with the Klenow fragment of Bacillus stearothermophilus DNA polymerase I (Bst). (Abstract shortened by UMI.).