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Mutation Detection Using RT-PCR-RFLP 319
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Mutation Detection Using RT-PCR-RFLP
Hitoshi Nakashima, Mitsuteru Akahoshi, and Yosuke Tanaka
1. Introduction
Genetic analysis by restriction fragment length polymorphism (RFLP) is one of
the most common methods used to examine nucleic acids for the presence of known
sequence variants. A segment that is to be searched for a mutation is amplified from
genomic DNA or cDNA, digested by the appropriate restriction enzyme, and then
separated by agarose gel electrophoresis. Although RFLP analysis is a highly sensitive
method that is easy to apply for the screening of known sequence variants, many
common polymorphisms are the result of single-base substitutions that fail to create
or remove any restriction site and, therefore, these cannot be readily typed by simple
polymerase chain reaction (PCR) and RFLP analysis. However, the use of a mismatch
PCR primer to artificially create a restriction site in the amplified product make it
possible to overcome this disadvantage (1,2). The mismatch primer contains a oneor
two-base mismatch near its 3′ end such that the amplified product incorporates or
removes a restriction site for the appropriate endonuclease in the presence of a base
substitution (3–5). The protocol for this method is the same as standard PCR.
2. Materials
1. RNA extracted from cells or tissues of interest.
2. Primers: stock solutions are at 100 µM in H 2 O; working solutions are at 10 µM in H 2 O.
The following primer pairs yield a 164-bp amplicon. The sites of mismatch are in capital
letters (3). Forward mismatch primer: 5′-ctc cta ccc ctt gtc atg cag gAt-3′; reverse primer:
5′-gtt aaa aca ggg acc tgt ggc atg-3′.
3. 10× PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3.
4. MgCl 2 (25 mM) in distilled water.
5. dNTPs (10 mM each of dATP, dGTP, dCTP, and TTP).
6. RNase inhibitor (20 U/µL).
7. Random hexamers (50 µM).
8. MuLV reverse transcriptase (50 U/µL).
9. AmpliTaq DNA polymerase (5 U/µL; Roche Molecular Systems, Inc. Branchburg, NJ).
10. Restriction enzyme FokI (10 U/µL) and digestion buffer.
11. DEPC-treated sterile H 2 O.
12. Sterile mineral oil.
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
319