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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