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1
Figure S1 . PNA-PCR/OCEAN method for K-ras
mutation detection. Assymetric PNA-PCR a PNA
probe complementary to the wild type gene
suppresses amplification of the codon 12 wild
type K-ras sequence and amplifies preferentially
mutant sequences. The PCR reaction is performed
using an imbalanced forwardreverse primer ratio
in order to preferentially amplify the strand
which is complementary to the OCEAN probes.
OCEAN steps 1-4 are carried out in a single
tube. Step 1 a stabilizing probe (anchor, in
dark gray) binds to the amplified strand of
K-ras step 2 a second probe (amplifier, in
light gray, carrying a Fluorescein and a Black
Hole Quencher, BHQ1) complementary to the mutant
gene recognizes and binds the duplex forming a
three-way junction. The resulting ternary
structure contains the recognition site for a
restriction endonuclease (RE). Step 3 The
endonuclease cleaves the amplifier. The cleavage
separates the fluorophore from the Black Hole
Quencher with consequent emission of
fluorescence. Step 4 the cleaved amplifier
dissociates, and the cycle repeats itself thus
resulting in a continuous signal amplification.
For each sample, 4 different OCEAN reactions are
performed, each containing a different labelled
amplifier, specific for the 4 most frequent
mutations of K-ras codon 12.
Asymmetric PNA-PCR/OCEAN for Kras mutation
detection
gene
mutated
mutated
mutated
mutated

gene
wild
type


wild
type


wild
type


wild
type

GGT
5

3

5

3

5

3

5

3

CCA
PNA
amplification
amplification
NO
amplification
NO
amplification
Step
4
Step
4
Step
4
Step
4
F
F
F
F
AAC
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
BHQ1
5

5

5

5

TTG
3

TTG
3

TTG
3

TTG
3

RE
F
Step
1
Step
1
Step
1
Step
1
AAC

TTG
TTG
TTG
TTG
TTG
TTG
TTG
TTG
5
3

3

3

3

3

3

3

3

Step 3
BHQ1
BHQ1
BHQ1
BHQ1
F
F
F
F
AAC
5

5

5

5

TTG
3

TTG
3

TTG
3

3

Step 2