Step
1
’ 5 Step1 Step4 BHQ1 TTG TTG F BHQ1 AAC 5’ 3’ 5’ 3’ F BHQ1 TTG 3’ Step1 Step4 BHQ1 TTG TTG F BHQ1 5’ 3’ 5’ 3’ BHQ1 TTG 3’ BHQ1 TTG 3’ gene mutated : gene wildtype : GGT 5’ 3’ CCA PNA: amplification NO amplification mutated : wildtype : 5’ 3’ Step1 Step4 BHQ1 TTG TTG F BHQ1 5’ 3’ 5’ 3’ BHQ1 TTG 3’ BHQ1 TTG 3’ Step1 Step4 BHQ1 TTG F BHQ1 5’ 3’ 5’ 3’ BHQ1 TTG 3’ BHQ1 TTG 3’ BHQ1 TTG 3’ mutated : wildtype : 5’ 3’ amplification NO amplification mutated : wildtype : 5’ 3’ GTT 5’ 3’ GTT 5’ 3’ GTT 5’ 3’ GTT 5’ 3’ Step 2 Step 3 RE Asymmetric PNA-PCR/OCEAN for Kras mutation detection AAC F AAC 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 forward:reverse 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 S S S
description
3. 3. 3. 3. ’. ’. ’. ’. 5. 5. 5. 5. ’. ’. ’. ’. GTT. GTT. GTT. GTT. S. S. S. Figure S1 . PNA-PCR/OCEAN method for K-ras mutation detection. - PowerPoint PPT Presentation
Transcript of Step
’5
Step 1
Step 4
BHQ1
TTG
TTG
F
BHQ1
AAC
5’3’
5’3’
F
BHQ1
TTG3’
Step 1
Step 4
BHQ1
TTG
TTG
F
BHQ1
5’3’
5’3’
BHQ1
TTG3’
BHQ1
TTG3’
gene mutated : gene wild type :
GGT5’ 3’CCAPNA:
amplification NO amplification
mutated : wild type :
5’ 3’
Step 1
Step 4
BHQ1
TTG
TTG
F
BHQ1
5’3’
5’3’
BHQ1
TTG3’
BHQ1
TTG3’
Step 1
Step 4
BHQ1
TTG
F
BHQ1
5’3’
5’3’
BHQ1
TTG3’
BHQ1
TTG3’
BHQ1
TTG3’
mutated : wild type :
5’ 3’
amplification NO amplification
mutated : wild type :
5’ 3’GTT5’ 3’GTT5’ 3’GTT5’ 3’GTT5’ 3’
Step 2
Step 3
RE
Asymmetric PNA-PCR/OCEAN for Kras mutation detection
AAC
FFFF
AAC
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 forward:reverse 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.
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