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Solid-Phase Minisequencing 361
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Analysis of Nucleotide Sequence Variations
by Solid-Phase Minisequencing
Anu Suomalainen and Ann-Christine Syvänen
1. Introduction
The Sanger dideoxynucleotide sequencing method has been simplified by a number
of methodological improvements, such as the use of the polymerase chain reaction
(PCR) technique for generating DNA templates in sufficient quantities followed
by affinity-capture techniques for convenient and efficient purification of the PCR
fragments for sequencing, or altenatively the use of cyclic Sanger sequencing reactions
that are easy to automate with laboratory robots, and the development of instruments
for automatic on-line analysis of fluorescent products of the sequencing reactions
(references to this book, solid phase sequencing, cyclic sequencing). Despite these
technical improvements, the requirement for gel electrophoretic separation remains
an obstacle when sequence analysis of large numbers of samples are needed, as in
DNA diagnosis, or in the analysis of sequence variation for genetic, evolutionary, or
epidemiological studies.
We have developed a method for analyzing DNA fragments that differ from each
other in one or a few nucleotide positions (1) denoted solid-phase minisequencing, in
which gel electrophoretic separation is avoided. Analogous to the methods for solidphase
sequencing of PCR products, the solid-phase minisequencing method is based
on PCR amplification using one biotinylated and one unbiotinylated primer, followed
by affinity-capture of the biotinylated PCR product on an avidin- or streptavidincoated
solid support. The nucleotide at the variable site is detected in the immobilized
DNA fragment by a primer extension reaction: A detection step primer that anneals
immediately adjacent to the nucleotide to be analyzed is extended by a DNA polymerase
with a single labeled nucleotide complementary to the nucleotide at the variable site
(Fig. 1). The amount of the incorporated label is measured, and it serves as a specific
indicator of the nucleotide present at the variable site.
We have used the solid-phase minisequencing method for detecting numerous
mutations causing human genetic disorders, for analyzing allelic variation in genetic
linkage studies, and for identifying individuals (2–4). The protocol described below
is generally applicable for detecting any variable nucleotide. The method suits well for
analyzing large numbers of samples because it comprises simple manipulations in
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
361