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MT-PCR for Microsatellite Analysis 295
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Combining Multiplex and Touchdown PCR
for Microsatellite Analysis
Kanokporn Rithidech and John J. Dunn
1. Introduction
An improved nonradioactive polymerase chain reaction (PCR)-based method for
simultaneous amplification of multiple loci of microsatellites has been developed as a
rapid way to screen for microsatellites (1). The approach, termed multiplex-touchdown
PCR (MT-PCR), is performed in a single PCR tube by combining touchdown (2–5) and
multiplex (6) PCR protocols. The touchdown format is used to improve the specificity
and the quality of amplification, that is, only DNA bands of an expected size are
present as the major PCR bands observed on the nondenaturing polyacrylamide gels,
thereby overcoming the presence of differently sized background bands (a ladder-like
problem). The multiplex strategy is used so that simultaneous amplification of multiple
microsatellite loci is achieved. In this chapter, we describe the MT-PCR strategy
that has been successfully used for simultaneous amplification of up to three mouse
microsatellites by choosing primer pairs with the corresponding touchdown-PCR
parameters. The MT-PCR is very useful for genotyping hybrid mice, provided the
allelic size difference between two parental genotypes is amenable to separation by
gel electrophoresis. In principle, this MT-PCR should be applicable to similar studies
in other species, including humans.
2. Materials
1. Sterile deionized water, for example, MilliQ water.
2. 10× PCR buffer (10 mM Tris-HCL, pH 8.3, 50 mM KCL).
3. 10 mM of each of dNTP (A,T,C, and G).
4. Microsatellite primers.
5. Taq polymerase.
6. MgCl 2 .
7. Template DNA (5 µg/mL, in sterile water).
8. Polyacrylamide (premixed 300.8 acrylamide:bisacrylamide, Owl Separation System
Portsmouth, NH).
9. Slab gel, for example, Mini-protean cell from Bio-Rad, large format single sided vertical
system (20 cm × 20 cm) from Owl separation System.
10. 1× TBE (Tris borate EDTA) gel running buffer.
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
295