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Detection of Nucleic Acids 65
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Technical Notes for the Detection of Nucleic Acids
John M. S. Bartlett
1. Introduction
In following any polymerase chain reaction (PCR)-based method, it is usual to
identify the products of the reaction by some form of detection system. The majority
of these still rely on size- and charge-based separation systems, although for some
quantitative PCR applications, either direct measurement of fluorescence or indirect
Enzyme-linked immunosorbent assay-based systems can be used. In this chapter, we
summarize some of the most common methods for detection of nucleic acids as a
handy reference for those seeking to validate their PCR reactions. Although there are
many variants of these techniques, we have confined our reporting to methods of which
we have direct experience and which offer broad applicability. Fluorescence detection
of quantitative real-time PCR requires specialist equipment, and we have therefore
omitted this from our discussions at present.
2. Gel Electrophoresis
By far the most common procedure for the analysis of nucleic acids is gel electrophoresis.
This is a highly flexible approach that provides information on the size of
the DNA molecule and under certain conditions can be used to discriminate different
sequences of the same size. Electrophoresis can also be used to separate and purify
nucleic acid fragments, to quantify allelic imbalances, etc. For DNA electrophoresis,
the most common supports used are agarose and polyacrylamide. These are highly
flexible because varying the concentration, and for polyacrylamide, the degree of
crosslinking, can markedly alter the size range which can be discriminated. In general,
polyacrylamide gels are more useful for separating smaller fragments of DNA (under
300–500 base pairs) and for applications where high resolution is required (such as
analysis of microsatellites) because they are capable of resolving size differences of
as little as 1 bp. Polyacrylamide gels can be run faster and at higher temperature
than agarose gels. However, acrylamide is a neurotoxin and presents a safety hazard.
Most laboratories now eschew the process of producing their own acrylamide solutions,
relying instead on commercially prepared materials to circumvent this hazard.
Polyacrylamide gels are also more difficult to pour and handle than agarose gels.
Furthermore, using polyacrylamide gels requires, in general, the use of labeled
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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