1 / 49

Alexander Lezhava Omics Sience Center RIKEN Yokohama Institute, Japan

Smart Amplification Process Introduction. Alexander Lezhava Omics Sience Center RIKEN Yokohama Institute, Japan. December 15 th 2009 , Tbilisi. Introduction

otto-sexton
Download Presentation

Alexander Lezhava Omics Sience Center RIKEN Yokohama Institute, Japan

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Smart Amplification Process Introduction Alexander Lezhava Omics Sience Center RIKEN Yokohama Institute, Japan December 15th 2009, Tbilisi

  2. 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.

  3. SmartAmp　（Smart Amplification Process) Nature Methods 3, 257-262 (2007)

  4. The Smart Amplification Process (SmartAmp) Traditional way DNA purification SNP detection DNA amplification SmartAmp DNA amplification SNP detection DNA purification Amplification = Signal Complete background suppression is essential

  5. 30分 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)

  6. Asymmetric primers and SNP detection points SNP detection points 3’ 5’ 5’ 3’ TP FP Asymmetric primers turnback primer (TP) and folding primer (FP)

  7. SmartAmp Amplification Overview Denatured genomic DNA 3’ 5’ 3’ 5’ ⅰ）Primer extension from the FP and TP. Strand displacement extension by the OP. Pathway C 3’ 3’ ⅱ）The FP and TP-linked strands are released and serve as a templates. 5’ 3’ ⅴ）Self-primed DNA synthesis. ⅴ）Self-primed DNA synthesis. Intermediate 1 3’ 5’ Intermediate 2 Pathway B Pathway A

  8. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ SmartAmp Amplification Overview Target DNA 3’ 5’ 3’ 5’ 3’ 3’ Denature

  9. SmartAmp Amplification Overview 3’ 5’ OP2 FP 3’ 5’ 3’ 5’ 3’ 3’ Generation of key intermediate 1

  10. SmartAmp Amplification Overview Key intermediate 1 TP OP1 3’ 5’ 5’ 3’ 5’ 3’ 3’ 3’ Generation of key intermediate 1

  11. SmartAmp Amplification Overview 5’ 3’ 5’ 3’ 3’ 3’ Key intermediate 1

  12. Key intermediate 1 SmartAmp Amplification Overview 5’ 3’ Pathway A

  13. SmartAmp Amplification Overview 3’ 5’ Pathway A

  14. SmartAmp Amplification Overview 3’ 5’ Pathway A

  15. SmartAmp Amplification Overview 5’ 3’ Pathway A

  16. IM1 equilibrium DNA Concatamers formed by SmartAmp 2% Agarose Gel Electrophoresis DNA yield is 100-1000x greater than PCR reaction Marker （ｂｐ） 500 400 331 242 190 147 110

  17. Aac DNA Polymerase Source: Alicyclobacillus acidocaldarius subsp. acidocaldarius JCM5260 • 60-68ºC optimal activity • Optimal pH 8.0-8.2 • Optimal cationic concentration = 8mM Mg2+ • Rapid rates of synthesis; typical of thermostabile enzymes 68 70 72 60 62 64 64 66 68 70 72 74 degree 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 1% agarose gel • Temperature stability of Aac DNA polymerase large fragment during assay on various temperature. • Commercially available DNA Polymerase I, large fragment • Aac DNA polymerase large fragment

  18. Key Background Suppression Strategy & IP SmartAmp’s unique background suppression technologies are the focus of multiple patent applications for isothermal amplification & molecular diagnostics. Mismatch binding protein（MutS) Asymmetric primer design

  19. MutS： 0μg 0.4μg 0.8μg 1.2μg MutS： 0μg 0.4μg 0.8μg 1.2μg Titration of MutS protein Titration performed on ~6,000 copies of human genomic DNA (lysed blood / unpurified DNA) Time(min) Time(min) Wt Template & Wt Primer Wt Template & Mut Primer SmartAmp Background Suppression by MutS • MutS protein binds mismatches and prevents amplification • Single nucleotide detection precision • SNP polymorphisms • Deletion detection • Background suppression depends on MutS titration and primer optimization

  20. Turn Back Primer (TP) Folding Primer (FP) Boost Primer (BP) - Wild Type - Boost Primer (BP) - Mutant - Outside Primer (OP1) Outside Primer (OP2) SmartAmp Reaction Assembly Master Mix Discrimination Primer Boost Primer (BP) Wild-type Assay Mutant (SNP) Assay

  21. SmartAmp Data Interpretation Heterozygous Homozygous wild-type Homozygous mutant Wild-type Assay Mutant (SNP) Assay

  22. ALDH2 – Aldehyde Dehydrogenase 2 Discrimination Primer is BP

  23. SmartAmp concordance with PCR (RFLP) data Genotype 1 2 Genome -ACACTGAAGTG--ACACTAAAGTG- 　　　　　 ↓　 　　　　　　　　　　　　　↓ Amino acid Glutamic acid Lysine 　　　　　　　　　　　 　　　　↓ ↓ Protein Active form ALDH2 Inactive form ALDH2 SMAP Method PCR (RFLP) Method • ALDH2 data on random population demonstrating 100% concordance of genotyping results with established PCR technology

  24. Mutation on EGFR and effect of Iressa EGFR Cytoplasm Cellular membrane Iressa Wilde Type Iressa Wilde type Mutant Inside of the cell Signal Signal proliferation Decreased proliferation Stop proliferation Cancer ガン

  25. Serious side effect -- Interstitial pneumonia Mutation on EGFR and effect of Iressa Science; 304: 1497-1500 (2004) New Eng. Med J. 350:2129-2139 (2004)

  26. Cancer Diagnosis – EGFR Example • SMAP may detect mutations that sequencing will miss • Results within 30 minutes from crude tumor lysate

  27. SMAP Wild-type assay Mutation assay Detection of trace amount of cancer cell cancer : normal＝1：9 Conventional methods failed to detect trace amount of cancer Sequencing pattern True Signal Error or Signal? You can detect trace amount of cancer cell by SMAP

  28. Clinical cases in NSCLC

  29. 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.

  30. Warfarin is the most widely prescribed anticoagulant for the treatment of thromboembolic disorders Clinically available warfarin VKORC1 (-1639G>A) S-Warfarin R-Warfarin Vitamin K oxide reductase Multiple CYP CYP2C9 Vitamin K 2,3-epoxide SNPs Exon 3, 430C>T for CYP2C9*2, Exon 7, 1075A>C for CYP2C9*3 g-glutamyl carboxylase Post-translational modification of blood coagulation factors II, VII, IX, X

  31. Detection of a desired gene from numerous family genes Alignment of CYP2C9*2 region against other family genes

  32. Detection of a desired gene from numerous family genes Template plasmid Red : CYP2C9*1 Blue : CYP2C9*2 Yellow : CYP2C19,18,8 Green : No Template CYP2C9*1 CYP2C9*2

  33. 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.

  34. 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. UGT1A1*28 WT: (TA)6TAA MT: (TA)7TAA 3’ 5´ 3´ 5´ 3´ 3’ Mutation Amplification TATATATATATA Wild type ATATATATATAT 3’ 5’ TATATATATATA × Competitive primer that prevents hybridization Mutant TATATATATATATA ATATATATATATAT 3’ 5’

  35. Detection of UGT1A1＊28 Wt/Wt (*1/*1) Wt/Mt (*1/*28) Mt/Mt (*28/*28) 4000 4000 4000 3000 3000 3000 FP(W) FP(M) Fluorescence (dR) Fluorescence (dR) 2000 2000 Fluorescence (dR) 2000 1000 1000 1000 FP(M) FP(W) 0 10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (min) Time (min) Time (min)

  36. 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.

  37. Nucleotide sequence of internal region of segment 6, Example. Conserved nucleotides are indicated in blue. Unstable nucleotides in red. …..CACCTTTTTCTAAGGACAATTCGATTaGGCTTTCCGCTGGTGGGGACATCTGGGTGACAAGAGAACCTTATGTGTCATGCGATCCTGACAAgTGTTATCAATTTGCCCTTGGaCAGGGAACAACACTAAACAACGtGCATTCAAATGACACAGTACaTGATAGGACCCCTTAtCGGACCCTATTGATGAATGAgttaGGTGT…..

  38. Detection of Tamiflu resistant mutation Neuraminidase activation site of H3N2

  39. Sample N1 against wild type and tamiflu mutant primer set Template: Swab RNA; sample #1 H1N1 sample #1 was amplified by wild type primer set Result: Sample #1 is H1N1 wild type and has no H274Y mutation. primer WT Mt

  40. 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 primer WT Mt

  41. Desk top type Traditional PCR thermal cycler Compact size type Isothermal fluorescent reader using card-type consumables Smart Amp (SMAP) Instruments

  42. BeckmanFX Roche LC480

  43. BeckmanFX Roche LC480

  44. New device – Handheld tube scanner SmartAmp reaction completed SmartAmp reaction Completed Diluted 1/2 Control tube

  45. Portable ESE-Quant TS (TubeScanner)- Simple configuration instead ofexpensive individual design Size ca. W: 20cm x D:15 cm x H: 7cm, Weight ca. 500gram Temperature control modules One or two dyes simultaneously Control and data evaluation software Configurable optical modules for any fluorescence dye

  46. Future Plans Device Application ■ 2-mm ultrathin disposable card using the chemical pocket stoveas heat source * Mockup ■Card-type device using a cell phone or a hand warmer as a heat source

  47. 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 have Been 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 most sample-preparation processes, which are the most time-consuming and troublesome steps in every diagnostics procedure.

  48. ALDH2 Typing Kit K-ras Mutation Detection Kit EGFR Mutation Detection Kit Warfarin Dosage Test Kit

  49. About us RIKEN carries out high level experimental and research work in a wide range of fields, including physics, chemistry, medical science, biology, and engineering, covering the entire range from basic research to practical application.

More Related