chapter  6
Sequencing the Single DNA Molecule
ByKenneth D. Weston, Markus Sauer
Pages 50

The Idea of Single-Molecule DNA Sequencing ...................................................107 Detection and Identification of Single Molecules

by Laser-Induced Fluorescence.........................................................................110 Identification of Single Molecules ........................................................................113 Incorporation and Degradation of Fluorescently Labeled Nucleotides

by DNA Polymerases and Exonucleases ..........................................................121 Single-Molecule DNA Sequencing in Hydrodynamically

Focused Sample Streams...................................................................................128 Single-Molecule DNA Sequencing in Microchannels

and Submicrometer Capillaries .........................................................................134 Future Prospects ....................................................................................................146 References..............................................................................................................148

The U.S. Human Genome Project was initiated in 1990 and was originally planned to last 15 years. Effective resources and technological advances have substantially accelerated the determination of the sequence of all 3 × 109 base pairs (bp) that make up human DNA and the identification of the approximately 30,000 genes in human DNA. Several types of genome maps have already been completed, and the first analysis of the working draft of the entire human genome sequence was published in February 2001.1,2 Although Sanger’s enzymatic chain termination method proved to be very reliable, the limited read length of <1000 bases per run requires the determination of overlapping subsequences to construct a “consensus” sequence of a larger DNA segment. Despite dramatic increases in speed over the past decade, existing procedures for sequencing remain labor-intensive and time-consuming. Improved sequencing methods are still needed to understand the function of each gene and genetic variations among cell types, individuals, and organisms. Furthermore, there is a growing interest in understanding the molecular basis of complex diseases and the variety of responses to drugs. The development of more effective