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

364 Suomalainen and Syvänen
detect a G, etc. (Amersham-Pharmacia Biotech; [ 3 H]dATP, TRK 633; dCTP, TRK 625;
dGTP, TRK 627; dTTP, TRK 576), stored at –20°C (see Note 3).
8. Scintillation fluid (for example Hi-Safe II, Wallac, Turku, Finland) stored at room
temperature (~20°C).
2.3. Primer Design
1. PCR primers: One PCR primer of each pair is biotinylated at its 5′ end during the synthesis
using a biotin-phosphoramidite reagent (for example Amersham–Pharmacia Biotech or
Perkin-Elmer–ABI; see Note 4).
2. The detection step primer for the minisequencing analysis is a 20-mer oligonucleotide
complementary to the biotinylated strand of the PCR product, designed to hybridize with
the 3′ end immediately adjacent to the variant nucleotide to be detected (see Fig. 1)
The minisequencing primer should be at least five nucleotides nested in relation to the
unbiotinylated PCR primer.
3. Methods
3.1. PCR for Solid-Phase Minisequencing Analysis
The PCR is performed according to routine protocols, except that the amount of
the biotin-labeled primer used is reduced not to exceed the biotin-binding capacity of
the microtiter well (see Note 1). For a 50-µl PCR, we used 10 pmol of biotin-labeled
primer and 50 pmol of the unbiotinylated primer. The PCR should be optimized (i.e.,
the annealing temperature and template amount) to be efficient and specific. To be
able to use [ 3 H] dNTPs, which are low-energy β-emitters, for the minisequencing
analysis, 1/10 of the PCR product should produce a single visible band after agarose
gel electrophoresis, stained with ethidium bromide. There is no need for purification
of the PCR product before the minisequencing analysis.
3.2. Solid-Phase Minisequencing Analysis
1. Affinity capture: Transfer 10-µL aliquots of the PCR product and 40 µL of the PBS/Tween
solution to two streptavidin-coated microtiter wells (see Note 5). Include a control reaction,
that is, a well with no PCR product. Seal the wells with a sticker and incubate the plate
at 37°C for 1.5 h with gentle shaking.
2. Discard the liquid from the wells and tap the wells dry against a tissue paper.
3. Wash the wells three times at room temperature as follows: pipet 200 µL of TENT solution
to each well, discard the washing solution and empty the wells thoroughly between the
washings (see Note 6).
4. Denature the captured PCR product by adding 100 µL of 50 mM NaOH to each well,
incubate at room temperature for 3 min. Discard the NaOH and wash the wells as in
step 3 above.
5. Prepare for each DNA fragment to be analyzed two 50-µL mixtures of nucleotide-specific
minisequencing solution, one for detection of the normal and one for the mutant nucleotide
(see Note 7). Mix 5 µL of 10× Taq DNA polymerase buffer, 10 pmol of detection step
primer, 0.2 µCi (usually equals to 0.2 µL) of one [ 3 H] dNTP, 0.1 U of Taq DNA polymerase,
and dH 2 O to a total volume of 50 µL. It is obviously convenient to prepare master mixes
for the desired number of analyses with each nucleotide.
6. Pipet 50 µL of one nucleotide-specific mixture per well, incubate the plate at 50°C for
10 min in a water bath or 20 min in an oven (see Note 8).
7. Discard the contents of the wells and wash them as in step 3.