mutation inserted by the oligo. However, the penultimate base
is designed to create a mismatch. Though weakened a bit for
annealing to its target, it still serves to generate a PCR signal
for the mutant DNA sequence. The same primer, however,
cannot easily recognize the wild type sequence as a result of a
2 base mismatch at its 3
0 end (i.e., due to the bp change
engineered at the penultimate position and the desired oligomediated bp change at the 3
0 end).
(a) This procedure can be time consuming though, as it may
require optimization of the PCR conditions for the primer
pairs described above to distinguish SNPs from wild type
sequences. However, if one uses a SNP oligo that created a
“bubble” at the annealing site to escape MMR
(as depicted in Fig. 2), one can take advantage of the
sequence change and design a primer that contains this
sequence at the 3
0 end. Such a primer would not easily
recognize the wild type sequence (with 4 out of 4 base pair
differences at the 3
0 end), but exactly match the SNP
sequence, giving a diagnostic PCR of greater specificity.
The same is true for differences that involve 3 out 4 bases.
If using this procedure, amplify by PCR a 300–500 bp
region containing the SNP site for each of the 24 candidates using such a SNP-specific primer. The presence of a
PCR fragment will distinguish which candidate contains
the SNP (along with the absence of such a fragment using
a wild type specific primer). Use Taq polymerase and 2 μL
of the heat-killed bacterial suspension in a 30-μL PCR
with the following program: step 1 (95
C—5 min);
step 2 (95
C—30 s); step 3 (58
C–61
C—30 s); step
4 (72
C—1 min); cycle through steps 2–4 (30Â); step 5
(72
C—5 min).
14. Even if the SNP is identified by PCR, sequencing of the SNP
region should still be done. When more than one candidate has
been identified by PCR, we have sometimes found (using the
sequencing chromatogram) that some of the candidates have
both the wild type and the modified base at the targeted
position. This may occur if the colony that was picked was
not well isolated, the colony grew as a mixture of cells, or
even by recombineering events occurring after the cells are
plated. It is important to check for mixed bases at the SNP
position, and if it is the only positive candidate, to then streak
out for single colonies and repeat the diagnostic PCR analysis.
15. A modified version of the procedure for obtaining heat-killed
cells for candidates containing the SNP is as follows. Single
colonies from the hygromycin plates are picked with sterile
loops and vigorously resuspended in 100 μL of PBS-tween in
1.5 mL screw-capped tubes. The cells are then
318
Kenan C. Murphy
is designed to create a mismatch. Though weakened a bit for
annealing to its target, it still serves to generate a PCR signal
for the mutant DNA sequence. The same primer, however,
cannot easily recognize the wild type sequence as a result of a
2 base mismatch at its 3
0 end (i.e., due to the bp change
engineered at the penultimate position and the desired oligomediated bp change at the 3
0 end).
(a) This procedure can be time consuming though, as it may
require optimization of the PCR conditions for the primer
pairs described above to distinguish SNPs from wild type
sequences. However, if one uses a SNP oligo that created a
“bubble” at the annealing site to escape MMR
(as depicted in Fig. 2), one can take advantage of the
sequence change and design a primer that contains this
sequence at the 3
0 end. Such a primer would not easily
recognize the wild type sequence (with 4 out of 4 base pair
differences at the 3
0 end), but exactly match the SNP
sequence, giving a diagnostic PCR of greater specificity.
The same is true for differences that involve 3 out 4 bases.
If using this procedure, amplify by PCR a 300–500 bp
region containing the SNP site for each of the 24 candidates using such a SNP-specific primer. The presence of a
PCR fragment will distinguish which candidate contains
the SNP (along with the absence of such a fragment using
a wild type specific primer). Use Taq polymerase and 2 μL
of the heat-killed bacterial suspension in a 30-μL PCR
with the following program: step 1 (95
C—5 min);
step 2 (95
C—30 s); step 3 (58
C–61
C—30 s); step
4 (72
C—1 min); cycle through steps 2–4 (30Â); step 5
(72
C—5 min).
14. Even if the SNP is identified by PCR, sequencing of the SNP
region should still be done. When more than one candidate has
been identified by PCR, we have sometimes found (using the
sequencing chromatogram) that some of the candidates have
both the wild type and the modified base at the targeted
position. This may occur if the colony that was picked was
not well isolated, the colony grew as a mixture of cells, or
even by recombineering events occurring after the cells are
plated. It is important to check for mixed bases at the SNP
position, and if it is the only positive candidate, to then streak
out for single colonies and repeat the diagnostic PCR analysis.
15. A modified version of the procedure for obtaining heat-killed
cells for candidates containing the SNP is as follows. Single
colonies from the hygromycin plates are picked with sterile
loops and vigorously resuspended in 100 μL of PBS-tween in
1.5 mL screw-capped tubes. The cells are then
318
Kenan C. Murphy