Alexander Lezhava Omics Sience Center
RIKEN Yokohama Institute, Japan
Smart Amplification ProcessIntroduction
December 15th 2009, Tbilisi
Introduction
Point of care of Molecular diagnostics is a rapidly growing area, whereby a broad range of advances in mutation detection technologies and their applications have appeared on the market. This includes developments in SNP genotyping systems applicable to point-of-care diagnostic testing, which have aided the validation of SNPs in the process of drug discovery.
We have developed a sensitive, accurate, rapid, and simple DNA amplification scheme that shows potential for various applications from pharmacogenomics-based drug discovery thru to point-of-care diagnostics.
Called the Smart Amplification Process (SmartAmp), the method employs a unique primer design and background suppression technology that can amplify target sequences from crude cell lysates without thermocycling.
SmartAmp ( Smart Amplification Process)
Nature Methods 3, 257-262 (2007)
Amplification = Signal
DNA purification
SmartAmp
DNA purification DNA amplification SNP detection
Traditional way
DNA amplificationSNP detection
Complete background suppression is essential
The Smart Amplification Process (SmartAmp)
Smart Amplification Process (SmartAmp) Extremely fast detection (15-30 min) Total background suppression; amplification = detection Simple data analysis; results are digital (no multiplex in current format) Primer design versatility Precision – single-nucleotide sequence specificity Sensitivity – at least equivalent to PCR Assay Robustness – genomic DNA amplification directly from blood High yield (1mg/100μl) Isothermal amplification (only two enzymatic components) Low energy requirements (lower cost instrumentation)
30 分
Asymmetric primers and SNP detection points
Asymmetric primers
3’5’
5’3’
SNP detection points
turnback primer (TP) and folding primer (FP)
FP TP
3’ 5’
3’5’
3’
3’
Pathway B
Pathway C
ⅰ) Primer extension from the FP and TP. Strand displacement extension by the OP.
ⅱ) The FP and TP-linked strands are released and serve as a templates.
Intermediate 2
ⅴ) Self-primed DNA synthesis. ⅴ) Self-primed DNA synthesis.5’
3’
3’ 5’
Denatured genomic DNA
Intermediate 1
Pathway A
SmartAmp Amplification Overview
SmartAmp Amplification Overview
5’
5’
3’
3’
5’
5’
3’
3’
Denature
3’ 5’
3’5’
3’
3’
Target DNA
SmartAmp Amplification Overview
5’ 3’
FP
OP2
3’ 5’
3’5’
3’
3’
Generation of key intermediate 1
SmartAmp Amplification Overview
3’5’
3’5’
3’
3’
TPOP1
Key intermediate 1
5’3’
Generation of key intermediate 1
SmartAmp Amplification Overview
Key intermediate 1
3’5’
3’5’
3’
3’
SmartAmp Amplification Overview
Pathway A
3’5’
Key intermediate 1
SmartAmp Amplification Overview
Pathway A
5’
3’
SmartAmp Amplification Overview
Pathway A
5’
3’
SmartAmp Amplification Overview
Pathway A
3’
5’
Marker
(bp)500400331242190147110
2% Agarose Gel Electrophoresis
IM1 equilibrium
DNA Concatamers formed by SmartAmp
DNA yield is 100-1000x greater than PCR reaction
S A B A B A B A B A B A B S A B A B A B A B A B A B
68 70 72 70 72 74 degree60 62 64 64 66 68
1% agarose gel
Aac DNA Polymerase Source: Alicyclobacillus acidocaldarius subsp. acidocaldarius JCM5260
Temperature stability of Aac DNA polymerase large fragment during assay on various temperature. A. Commercially available DNA Polymerase I, large fragment B. Aac DNA polymerase large fragment
60-68ºC optimal activity Optimal pH 8.0-8.2 Optimal cationic concentration = 8mM Mg2+
Rapid rates of synthesis; typical of thermostabile enzymes
Key Background Suppression Strategy & IP
Mismatch binding protein ( MutS)
Asymmetric primer design
SmartAmp’s unique background suppression technologies are the focus of multiple patent applications for isothermal
amplification & molecular diagnostics.
MutS protein binds mismatches and prevents amplification
Single nucleotide detection precision SNP polymorphisms Deletion detection
MutS : 0μg 0.4μg 0.8μg 1.2μg
MutS : 0μg 0.4μg 0.8μg 1.2μg
Time(min) Time(min)
Titration of MutS protein
Wt Template & Mut PrimerWt Template & Wt Primer
Titration performed on ~6,000 copies of human genomic DNA (lysed blood / unpurified DNA)
Background suppression depends on MutS titration and primer optimization
SmartAmp Background Suppression by MutS
SmartAmp Reaction Assembly
Boost Primer (BP) - Wild Type -
Boost Primer (BP) - Mutant -
Turn Back Primer (TP)
Folding Primer (FP)
Outside Primer (OP1)
Outside Primer (OP2)
Boost Primer (BP)
Discrimination Primer
Wild-type Assay Mutant (SNP) Assay
Master Mix
SmartAmp Data Interpretation
Wild-type Assay Mutant (SNP) Assay
Heterozygous Homozygous mutantHomozygous wild-type
ALDH2 – Aldehyde Dehydrogenase 2
Discrimination Primer is BP
SmartAmp concordance with PCR (RFLP) data
1/1 1/2 2/2
1/1 32 0 0
1/2 0 18 0
2/2 0 0 3
SMAP MethodP
CR
(R
FLP
) M
etho
d
• ALDH2 data on random population demonstrating 100% concordance of genotyping results with
established PCR technology
Genotype 1 2 Genome -ACACTGAAGTG- -ACACTAAAGTG- ↓ ↓Amino acid Glutamic acid Lysine ↓ ↓ Protein Active form ALDH2 Inactive form ALDH2
EGFR
Signal
IressaWilde type
proliferation
Iressa
Signal
Decreased proliferationStop proliferation
Mutation on EGFR and effect of Iressa
Wilde Type
Mutant
ガン Cancer
Cellular membrane
Inside of the cell
Cytoplasm
Science; 304: 1497-1500 (2004)New Eng. Med J. 350:2129-2139 (2004)
Serious side effect-- Interstitial pneumonia
EGFR gene Effective Non-effective
Mutant 13 0
Wilde type 1 11
Mutation on EGFR and effect of Iressa
Cancer Diagnosis – EGFR Example
SMAP may detect mutations that sequencing will miss
Results within 30 minutes from crude tumor lysate
Detection of trace amount of cancer cell
Sequencing pattern
Wild-type assay
Mutation assay
SMAP
Error or Signal?
You can detect trace amount of cancer cell by SMAP
True Signal
cancer : normal = 1 : 9Conventional methods failed to detect trace amount of cancer
CaseNumber
Age Histotype Stage Detection by sequencing By SMAP
DeletionType
5 73 F ADC ΙAexon 19 deletion 2235-
2249exon 19 deletion 2235-
2249DE-A
8 61 M ADC ⅠBexon 19 deletion 2239-
2247 2248G>Cexon 19 deletion 2239-
2247 2248G>CDE-D
17 54 F ADC ⅢBexon19 deletion 2239-
2248 2252-2256exon19 deletion 2239-
2248 2252-2256DE-G
22 72 M ADC ⅢAexon19 deletion 2236-
2250exon19 deletion 2236-
2250DE-B
30 64 F ADC ⅢAexon 19 deletion 2235-
2249exon 19 deletion 2235-
2249DE-A
3 68 M ADC ⅢA Wild type exon 21 L858R
14 83 F ADC ⅠB exon 21 L858R exon 21 L858R
16 74 F ADC ⅠA exon 21 L858R exon 21 L858R
26 52 F ADC ⅢA exon 21 L858R exon 21 L858R
2860 M ADC ⅢB exon 21 L858R exon 21 L858R
Clinical cases in NSCLC
Rapid SNP detection of the cytochrome P450 (CYP) 2C9 and the vitamin K oxide reductase (VKOR) gene for the
warfarin dose adjustment.
Warfarin is the most widely prescribed anticoagulant for the treatment of thromboembolic disorders.
The genetic polymorphism of the cytochrome P450 (CYP) 2C9*2, CYP2C9*3 and the vitamin K oxide reductase (VKOR) -1639G>A greatly impact the maintenance dose for the drug, warfarin.
Pre-screening patients for these genotypes, prior to prescription of the drug will facilitate a more rapid individualized determination of the proper maintenance dose for a patient, minimizing risk for adverse reaction and reoccurrence of thromboembolic episodes.
SmartAmp assay system was developed for above-mentioned SNP detection. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 40 minutes.
Clinically available warfarin
R-Warfarin
Multiple CYP
Vitamin K oxide reductase
g-glutamyl carboxylase
Post-translational modification of blood coagulation factors II, VII, IX, X
Vitamin K 2,3-epoxide
Warfarin is the most widely prescribed anticoagulant for the treatment of thromboembolic disorders
VKORC1
SNPs Exon 3, 430C>T for CYP2C9*2,Exon 7, 1075A>C for CYP2C9*3
(-1639G>A)
S-Warfarin
CYP2C9
Alignment of CYP2C9*2 region against other family genes
Detection of a desired gene from numerous family genes
Template plasmidRed : CYP2C9*1Blue : CYP2C9*2
CYP2C9*1 CYP2C9*2
Yellow : CYP2C19,18,8Green : No Template
Detection of a desired gene from numerous family genes
Possible pathways of irinotecan metabolism. Irinotecan (CPT-11) can be converted into the active metabolite SN-38 by carboxylesterases (CES) outside or inside the cell.
3’
3´
5´3´
5´
Mutation 3’
UGT1A1*28 WT: (TA)6TAA MT: (TA)7TAA
3’ 5’ Wild type Amplification
ATATATATATATTATATATATATA
3’ 5’
×ATATATATATATAT
TATATATATATA
TATATATATATATA Mutant
Competitive primer that prevents hybridization
The dinucleotide - repeat sequences (5-8 repeats) in the promoter for uridine 5´-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) has an influence on glucuronidation of SN-38, the active metabolite of irinotecan. Patients with seven repeat sequences have a fourfold relative risk of experiencing severe toxicity after treatment with irinotecan, including grade III/IV diarrhea and leucopenia, compared with patients with six repeat sequences.
Wt/Wt(*1/*1)
Wt/Mt(*1/*28)
Mt/Mt(*28/*28)
FP(W)
FP(M)
FP(M)
FP(W)
0
1000
2000
3000
4000
10 20 30
Flu
ores
cenc
e (d
R)
40 50 60Time (min) 0
1000
2000
3000
4000
10 20 30
Flu
ores
cenc
e (d
R)
40 50 60Time (min)
1000
2000
3000
4000
Flu
ores
cenc
e (d
R)
0 10 20 30 40 50 60Time (min)
Detection of UGT1A1 * 28
RT-SmartAmp and Detection of Tamiflu Resistant Influenza A H3N2 and H1N1 Viruses
The use of influenza virus neuraminidase (NA)-specific inhibitors, is rapidly increasing in Japan.
Neuraminidase mutations in Influenza viruses from clinical patients have been reported.
Tamiflu resistant influenza A and B viruses have circulated in humans.
RT-SmartAmp was applied on single mutation detection in influenza A H3N2 viruses, which are leading to the Tamiflu resistance.
Nucleotide sequence of internal region of segment 6, Example. Conserved nucleotides are indicated in blue. Unstable nucleotides in red.
…..CACCTTTTTCTAAGGACAATTCGATTaGGCTTTCCGCTGGTGGGGACATCTGGGTGACAAGAGAACCTTATGTGTCATGCGATCCTGACAAgTGTTATCAATTTGCCCTTGGaCAGGGAACAACACTAAACAACGtGCATTCAAATGACACAGTACaTGATAGGACCCCTTAtCGGACCCTATTGATGAATGAgttaGGTGT…..
Neuraminidase activation site of H3N2
119E primer set + MutS 4ug 119vprimer set + MutS 4ug119E 119v
292R primer set with CP (10 times) 292k primer set with CP (5 times)292R 292k
294N primer set 294s primer set with CP (10 times)294N 294s
Detection of Tamiflu resistant mutation
WTMt
primer
Template: Swab RNA; sample #1
H1N1 sample #1 was amplified by wild type primer set
Sample N1 against wild type and tamiflu mutant primer set
Result: Sample #1 is H1N1 wild type and hasno H274Y mutation.
WTMt
primer
Sample N2 against H1N1 wild type and H274Y mutant primer set
Template: Swab RNA; sample #2
H1N1 sample #2 was amplified by mutant H274Y primer set only
Result: Sample#2 is H1N1 Tamiflu resistant virus
Smart Amp (SMAP) Instruments
Traditional PCR thermal cycler
Isothermal fluorescent reader using card-type consumables
Desk top type
Compact size type
BeckmanFX Roche LC480
BeckmanFX Roche LC480
New device – Handheld tube scanner
Control tube
SmartAmp reaction completed
SmartAmp reaction CompletedDiluted 1/2
Portable ESE-Quant TS (TubeScanner)- Simple configuration instead of expensive individual design
Size ca. W: 20cm x D:15 cm x H: 7cm, Weight ca. 500gram
Configurable optical modules for any fluorescence dye
One or two dyes simultaneouslyTemperature control modules
Control and data evaluation software
Future Plans
Device
■ Card-type device using a cell phone or a hand warmer as a heat source
■ 2-mm ultrathin disposable card using the chemical pocket stove as heat source
* Mockup
Application
Conclusion
SmartAmp is a rapid mutation detection method that accomplishes perfect background suppression using new polymerase with strand displacement activity, asymmetric primer design and mismatch-binding protein
Battery-operated, handheld, and mobile diagnostic testing platforms haveBeen built and can provide sensitive, accurate, and specific results as well as Rapid turnaround time, operational and physical robustness, and affordability.
The assays work directly from clinical samples such as urine and blood.
Due to their mobility, the platforms avoid sample storage and transportation.
And due to their ability to work directly from clinical samples, they avoid mostsample-preparation processes, which are the most time-consuming andtroublesome steps in every diagnostics procedure.
ALDH2 Typing Kit
EGFR Mutation Detection Kit Warfarin Dosage Test Kit
K-ras Mutation Detection Kit
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