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

Inosine-Containing Primers 371
2. Transfer 15 to 16 µL of the above labeled mixture to a new tube. Avoid carryover of any
mineral oil. This can be done easily by putting the pipetting tip directly below the oil
without touching the wall of the tube.
3. Optional (see Note 5): Load 1 to 2 µL with 1 µL of gel-loading buffer to a sequencing
gel to check the labeling efficiency.
4. Termination PCR mix: For each of the labeled mixes, prepare four tubes labeled as “G,”
“A,” “T,” and “C.” To each of the tubes, add 4 µL of termination mix G, A, T, or C (this can
be done toward the end of label-PCR procedure). Add 3.5 µL of the label mix to each of
the tubes. Cover the termination PCR mix with 8 to 10 µL of mineral oil.
5. Termination PCR: Cycle between 95°C for 30 s and 72°C for 90 s (see Note 6).
6. While the termination PCR is under way, prepare four clean 0.5-mL tubes labeled G, A, T,
or C. To each of them add 4 µL of stop/gel-loading solution.
7. Transfer 6 to 7 µL of termination mix to these tubes with the stop/loading solution.
Avoid carryover of mineral oil. Mix and spin down briefly and store at –20°C (good for
up to 1 mo). These samples are ready for the sequencing gel (use 3 µL to load a gel).
See Chapter 51.
8. Sequencing results: run sequencing gel, perform autoradiography, and read the sequence
(see Note 7).
4. Notes
1. Cetus Perkin–Elmer thermal cycler Model 9600 also may be used. If it is, use 0.1-mL
tubes; no mineral oil on the top of the reaction solution is needed. The PCR program
should be adjusted accordingly in the procedure.
2. Other methods of DNA preparation are also acceptable as long as “clean” DNA is
obtained.
3. Other {a- 35 S-labeled nucleotides may also be used, but the label mix must be changed
accordingly. The concentration of the stock of degenerate primers in the reaction is
dependent on the degree of degeneracy. In our case, the stock concentration of our >100∞
degenerate primer was 200 µM. Because radioactive 35 S is used for these experiments,
always be careful and follow the safety operation procedure for your institute. Check
with your radiation safety officer for the authorized amount of radioactivity that you can
handle at any one time.
4. Depending on the sequencing primer, the annealing/elongation temperature or time may
have to be optimized to give proper primer extension and labeling. The purposes of the
label PCR is to have a sufficient amount of specific primer in the primer mix to anneal to a
specific site on the DNA template and to extend the annealed primer for a limited nucleotide
length with Taq DNA polymerase. The first purpose can be achieved by choice of an optimal
annealing temperature and/or time. In our case (200 pmol of the 20 mer with inosine and
a degeneracy of more than 100∞), we used 52°C and 30 s. Depending on circumstances,
this temperature and the annealing time may need to be adjusted. The method of generating
a limited elongated primer plus labeling of the primer is accomplished by using a shorter
annealing/elongation time at a suboptimal temperature (for Taq activity), but still a stringent
temperature for annealing and a low dNTP concentration. In this way, it is not necessary to
know the sequence of the downstream flanking region of the sequencing primer.
5. Loading 1 to 2 µL of labeled mix to run a gel to check that the length of primer extension
and label efficiency is optional. This can be run along with the sequencing sample after
all the reactions are finished. Using our p450 degenerate sequencing primers under these
labeling PCR conditions, we obtained an average primer extension of 15 to 25 bp.
6. The temperature used for both the annealing of labeled/extended primers to the DNA
template for the elongation/termination reaction was 72°C. Only the prelabeled and pre-