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Direct Automated Cycle Sequencing 341
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Preparation and Direct Automated Cycle Sequencing
of PCR Products
Susan E. Daniels
1. Introduction
The polymerase chain reaction (PCR) is well known for being a rapid and versatile
method for the amplification of defined target DNA sequences. This technique can be
applied to a variety of research areas, such as the identification and typing of single
nucleotide substitutions of DNA sequence polymorphisms, and genetic mapping (1–4).
Since the introduction of PCR (5), a variety of methods for sequencing PCRgenerated
fragments have been described. These are usually based on the Sanger
chain-terminating dideoxynucleotide sequencing (6) rather than the Maxam and Gilbert
chemical cleavage method (7). Manual dideoxy sequencing methods are labor intensive,
time-consuming, involve radioisotopes, and have limitations in sequence ordering.
However, a technique combining the PCR and dideoxy terminator chemistry simplifies
the process of sequencing and is known as cycle sequencing (8). Automated or
fluorescent DNA sequencing is a variation of the traditional Sanger sequencing
using the cycle sequencing methodology, where fluorescent labels are covalently
attached to the reaction products and data are collected during the polyacrylamide
gel electrophoresis.
The introduction of fluorescently labeled dideoxynucleotides as chain terminators
presented the opportunity for the development of reliable cycle sequencing for PCR
products. The sequencing reaction with the dye terminators is performed in a thermal
cycler, and each of the four dideoxynucleotide triphosphates (ddNTPs) is labeled
with a different fluorescent dye. This allows the four chain extension reactions to be
conducted within a single tube, sparing considerable labor (9,10). The use of labeled
chain terminators allows flexibility of sequencing strategy because the same primers
can be used in the sequencing reaction. This eliminates the time and expense associated
with a separate set of modified DNA sequencing primers and is well suited to high
throughput sequencing.
Using this method, it is possible to amplify a target DNA sequence, purify the
resulting fragment, and obtain sequencing data within 24 h. Also, it has been used
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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