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Mutation and Polymorphism Detection 287
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Mutation and Polymorphism Detection
A Technical Overview
Joanne Edwards and John M. S. Bartlett
1. Introduction
Analysis of DNA variation (polymorphism and mutations) is one of the most
common challenges faced by molecular biologists. Studies of polymorphisms and
mutations as molecular markers of or underlying causes of disease have confirmed
the importance of mutation and polymorphism detection. With mutation detection
currently being so important for the study of genetic diseases, gene discovery, and
solving problems of basic biology, there is a large demand for quick and relatively
cheap methods for mutation detection. Therefore, many different methods have been
developed for detecting new mutations and screening populations for known mutations
or polymorphisms. Traditional mutation detection systems, such as restriction fragment
length polymorphism and denaturing gradient gel electrophoresis, have their limitations.
With restriction fragment length polymorphism, the mutational event needs to either
create or destroy a restriction site (1). With denaturing gradient gel electrophoresis,
although a change at a restriction digest site is not required, this method may only
detect about 50% of possible mutations and polymorphisms (1). The use of polymerase
chain reaction (PCR)-based mutation and polymorphism detection systems
increase the sensitivity and accuracy of the screening methods. This chapter will introduce
some of the available detection methods and discuss the problems associated
with these techniques.
2. Detection Methods
2.1. Detection of Single Nucleotide Polymorphisms (SNPs)
2.1.1. Single-Strand Conformation Polymorphism (SSCP)
SSCP can detect up to 90% of single base changes and relies on the different
mobilities of DNA strands containing single bp differences when run on a denaturing
polyacrylamide gel (2). SSCP analysis is widely used to screen large numbers of samples
for mutations. If a mutation is detected, direct sequence analysis is often then used to
determine the exact location and base change of the mutation. SSCP can be performed
using either fluorescent or radioactive technology (2,3). The advantages of fluorescent
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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