Next Generation DNA Sequencing and PCR Protocols & Manipulation

Nucleic acid sequencing is a method for determining the exact order of nucleotides present in a given DNA or RNA molecule. In the past decade, the use of nucleic acid sequencing has increased exponentially as the ability to sequence has become accessible to research and clinical laboratories all over the world. The first major foray into DNA sequencing was the Human Genome Project, a $3 billion, 13-year-long endeavor, completed in 2003. The Human Genome Project was accomplished with first-generation sequencing, known as Sanger sequencing. Sanger sequencing (the chain-termination method), developed in 1975 by Edward Sanger, was considered the gold standard for nucleic acid sequencing for the subsequent two and a half decades (Sanger et al., 1977). Following the human genome project, 454 was launched by 454 in 2005, and Solexa released Genome Analyzer the next year, followed by SOLiD (Sequencing by Oligo Ligation Detection) provided from Agencourt, which are three most typical massively parallel sequencing systems in the next generation sequencing (NGS) that shared good performance on throughput, accuracy, and cost compared with Sanger sequencing. These founder companies were then purchased by other companies: in 2006 Agencourt was purchased by Applied Biosystems, and in 2007, 454 was purchased by Roche, while Solexa was purchased by Illumina. After years of evolution, these three systems exhibit better performance and their own advantages in terms of read length, accuracy, applications, consumables, manpower requirement and informatics infrastructure, and so forth. This book will compile and compare these major advances in DNA sequencing automation and PCR methodology.