QX200 Droplet Digital PCR System의 소개 - …

1 QX200TM Droplet DigitalTM PCR System 1. Digital PCR Digital PCR PCR 3 . PCR 1 , 2 PCR (Real-time PCR) , 3 PCR PCR . 1 PCR agarose gel , . 2 PCR Real-time PCR Ct value . Real-time PCR , PCR . Digital PCR . 1. PCR 2. Droplet DigitalTM PCR ? Droplet DigitalTM PCR System 20ul PCR 2 Droplet , target DNA.

2 Droplet target DNA positive Droplet (1) negative Droplet (0) Digital signal , target DNA copy ul Copy . Bulk (20ul) Droplets (1nl x 20,000) one measurement many thousands of discrete measurements Real-time PCR Digital PCR 2. PCR Real-time PCR Digital PCR (Partitioning) 3. QX200TM Droplet DigitalTM PCR Workflow probe (FAM, HEX/VIC) EvaGreen PCR 20ul.

3 PCR Droplet Generator 2 2 droplets . 8 . droplets 96-well PCR plate , plate 96-well PCR PCR . PCR plate Droplet Reader . Reader QuantaSoft 96 positive negative Droplet , copy . 3. Droplet Digital PCR (1) Prepare ddPCR reaction mix - DNA template 20ul ddPCR (2) Generate droplets- Droplet generation oil 70ul PCR 20ul well QX200 Droplet Generator 2 Droplet (3) Perform PCR - droplets 96-well PCR plate , thermal cycler PCR (4) Read and analysis results PCR 96-well PCR plate QX200 Droplet Reader QuantaSoft Droplet FAM HEX copy 4.

4 Automated Droplet Generator PCR oil , droplets 96-well PCR plate . 96 hands-on 5 high-throughput 4. QX200TM Droplet DigitalTM PCR QX200TM Droplet DigitalTM PCR QuantaSoft , Droplet , . , FAM HEX 1D 2D-plot negative, positive cluster , negative positive Droplet threshold . Negative positive Droplet signal concentration, copies/ul . FAM/HEX ratio , Droplet event.

5 (A) (B) 5. QuantaSoft (A)2D-plot (Black dots: FAM-/HEX-, Blue dots: FAM+/HEX-, Brown dots: FAM+/HEX+, Green dots: FAM-/HEX+) (B) concentration . (FAM, Blue square: target gene, 2-fold serial dilution / HEX, Green square: reference house-keeping gene) 5. Droplet Digital PCR 1) RT-PCR 1,000 - 2) Standard curve 3) precision accuracy - PCR inhibitor - PCR - 4) RT-PCR , ddPCR copy (1 copy resolution) 5) IHC, FISH ( ddPCR ) 6.

6 QX200TM Droplet DigitalTM PCR 1) background DNA - FFPE HER2 (Method / 2013 / Nicholas J. Heredia et al.) - cell free plasma DNA EGFR mutation (Clinical Cancer Research / 2014 / Geoffrey R. Oxnard et al.) - circulating tumor cell circulating tumor DNA (Cancer discovery / 2014 / Daniel A. Haber et al.) - plasma cancer DNA (Clinical Cancer Research / 2014 / Julia A. Beaver et al.) - NRAS BRAF RAF MEK inhibition (Cancer discovery / 2014 / Abdel-Wahab O et al.) 2) Stem cell single cell - pre-leukaemic haematopoietic stem cell (Nature / 2014 / Liran I.

7 Shlush et al.) - Antibiotic selection hiPSC (Nature methods / 2014 / Yuichiro Miyaoka et al.) - miRNA137 (Stem cell research / 2013 / Ke Jiang et al.) 3) - (Science / 2014 / Brett E. Johnson et al.) - Cytomegalovirus ddPCR (Journal of clinical microbiology / 2014 / Ruth Hall Sedlak et al.) - (Genetics in medicine / 2014 / Wei Gu et al.) - HAM/TSP T viral mutation (Journal of neurovirology / 2014 / Giovanna S. Brunetto et al.) 4) - High throughput DNA copy number (Analyticalchemistry / 2011 / Benjamin J.

8 Hindson et al.) (Analyticalchemistry / 2011 / Leonardo B. Pinheiro et al.) - miRNA real-time PCR Digital PCR (Nature methods / 2013 / Christopher M Hindson et al.) - plasma miRNA (Biomarker insights / 2013 / Jie Ma et al.) - HIV DNA (PLOS One / 2013 / Matthew C. Strain et al.) 5) NGS validation library - haplotype NGS validation (Nature genetics / 2012 / Linda M Boettger et al.) - Illumina TruSeq library Ion Torrent library (BioTechniques / 2013 / Matthew T. Laurie) 7. R&D 100 Award Frost and Sullivan Digital PCR , QX200TM Droplet DigitalTM PCR System 2011 150.

9 2013 qPCR Digital PCR , QX200TM Droplet DigitalTM PCR probe EvaGreen dye . SYBR Green Digital PCR . Bio-Rad validation CNV Analysis kit Mutation Detection kit , NGS library quantification kit , Digital PCR . , QX200TM Droplet DigitalTM PCR . BMS Bio-Rad Bio-Rad , . : ( ) , BioScience PM