In essence, reading the base sequence of any particular segment of DNA using Sanger's dideoxynucleotide chain termination method is achieved through the creation of sequential fragments that match the chain to be sequenced, each fragment being one base longer than the previous one. In four separate reactions, each containing a different terminator dideoxy base (A, G, T, C), one of the four radiolabeled dideoxynucleotides is incorporated into the growing chain of DNA during enzymatic synthesis; its incorporation stops further chain growth resulting in a collection of fragments. The fragments differ in length and they all end in the dideoxynucleotide incorporated during DNA synthesis. The fragment collection that results is then separated according to length by gel electrophoresis. By determining the identity of the final radiolabeled nucleotide in each fragment, the entire sequence could be reconstructed. Manual performance of this process by an experienced investigator permitted the determination of up to about 5000 bases in a week.
In 1985, Hood and colleagues developed a strategy by which the radiolabeled dideoxynucleotides were replaced with fluorescent dyes covalently attached to the oligonucleotide primer used during DNA synthesis; a different colored dye specific for the bases A, G, T, C was used for each synthetic reaction. The reaction mixtures were combined, coelectrophoresed down a single polyacryl-amide gel tube, and the fluorescent bands of DNA were separated near the bottom of the tube. The fluorescent bands within the gel were detected by the fluorescence emission spectra excited by an argon laser at four different wavelengths. The sequence was acquired directly by computer. Specialized chemistry that was developed for attaching the dyes to the DNA primer, for fluorescence detection, and for data analysis demonstrated that automated DNA sequence analysis was a practical goal.11 This first step toward automation eliminated much of the labor associated with DNA analysis and demonstrated that an investigator could read more sequences in a day than could be read manually in an entire week. On the basis of this work, LeRoy Hood in collaboration with colleagues at Applied Biosystems built the first commercial instrument, ABI Model 370, for DNA sequence analysis.
Soon after, a number of innovations and refinements to automatic DNA sequencing were introduced. Simpler chemistry for fluorescent labeling of sequence terminator dyes was devised and with this innovation, the entire sequencing reaction could be performed in a single tube. More powerful computers, increased gel capacity, improvement of the optical systems, and more sensitive fluorescent dyes increased the capacity of the instrument so that the ABI PRISM
Model 377 sequencer introduced in 1995 could sequence more than 19,000 bases per day. Owing to the dependence on slab gel electrophoresis, however, the capacity was limited, time consuming, and cumbersome. Subsequently, this limitation was addressed in the newer machine, the ABI Model 3700 Automated Sequencer, by using capillary electrophoresis for separating DNA fragments. And the technology for DNA sequence analysis has continued to advance. According to Applied Biosystems, the 3730xL sequencer can process 96 capillaries, each with the capacity to call 900 bases, in 3 hours with a 1% error rate.133
Two attempts to reduce the time and expense of DNA sequence analysis have recently been reported. The core technology of base-calling from emission wavelength, which had not changed appreciably since Hood's research in 1986,11 had some disadvantages limiting sensitivity and application to multicomponent systems. Metzker and colleagues presented an approach called pulsed multiline excitation (PME) that uses four different lasers matched to four different dyes to improve sensitivity and broaden application.139 The higher sensitivity translates into significantly enhanced signal quality that in turn would decrease the cost of fluorescent dye reagents as illustrated in the application to primary DNA sequencing data. In another effort, Margulies and associates searched for alternative methods to reduce the time and cost of DNA sequence analysis. They described a scalable, highly parallel sequencing system with greater throughput than state-of-the-art capillary electrophoresis instruments. They carried out the shotgun sequencing and assembly of the Mycoplasma genitalium genome with 96% coverage of 99.96% accuracy in one 4-hour run of the machine. Their apparatus used a new fiberoptic slide of individual wells and could sequence 25 million bases, at 99% or better accuracy, in 4 hours.140 Shendure and colleagues described a technology that converts an inexpensive epifluorescence microscope to rapid nonelectrophoretic DNA sequencing automation.6 They used this technology to resequence an evolved strain of E. coli at a cost of less than one-ninth that of conventional sequencing and with less than one error per million bases.
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