John M. S. Bartlett.pdf - Bio-Nica.info

MT-PCR for Microsatellite Analysis 297
3.3. Step 3: Touchdown
A typical PCR protocol for the initial Touchdown cycle is denaturation at 96°C for
30 s, annealing at 65°C for 30 s, and extension at 72°C for 30 s.
3.4. Step 4: Standard PCR Cycles
After 5 to 10 touchdown cycles (depending on the targeted annealing temperature),
20 standard PCR cycles are performed under the following conditions: denaturation at
96°C for 30 s, annealing at 55°C (or 60°C) for 30 s, extension at 72°C for 30 s, and
final extension at 72°C for an additional 5 min (see Note 3).
3.5. Step 5: Gel Electrophoresis
Typically, 6 and 10% nondenaturing polyacrylamide gels are used for separating
microsatellites with allelic size differences of >12 bp and 20 cycles) results in deterioration of
the PCR specificity leading to an increase in the background of spurious bands. Under
the proper conditions, several amplicons from one round of MT-PCR can be analyzed
simultaneously and each amplicon can be identified unambiguously on the same lane of
nondenaturing PAGE. By choosing primer pairs with the corresponding T-PCR parameters,
the MT-PCR is highly efficient for concurrent identification of three microsatellite loci
(Fig. 1, lane 1).
3. A soak file at 4°C can be set after the final extension step.
4. A Mini-protean cell from Bio-Rad is used for the 6% nondenaturing polyacrylamide gel.
A large format single sided vertical system (20 cm × 20 cm) from Owl separation System
is used for the 10% nondenaturing polyacrylamide gel.
5. Beside speed and ease, an additional advantage of MT-PCR is that it is notably useful
when only small quantities of DNA template are available. Amounts of DNA template
as low as 10 ng can be used in one simultaneous reaction with three different pairs of
primers. DNA samples from different tissues of the mouse, that is, bone marrow cells,
spleen, liver, and tail, have been used successfully as templates in MT-PCR. Normally,
the simple salting out procedure for isolating genomic DNA described by Miller et al. (8)
routinely gives, in our laboratory, high-quality DNA templates from these tissues. The
only drawback to the use of MT-PCR for the genotyping of microsatellites is that each
protocol depends largely on primer compatibility, that is, having similar reaction kinetics
(amenable to the same T-PCR protocol).