qPCR
technique to routine applications is hampered by the rather high cost per sample analysed. This is about to change. Since digital PCR is an end-point technique that does not require continuous moni- toring of product formation, the arena is open for other platforms. A spinning disc of inexpensive plastic with microfluidic architecture has been designed that passively compartmentalises a sam- ple into 1,000 nanolitre-sized wells by centrifuga- tion. A rapid air thermocycler is used for PCR and a CCD camera to acquire a fluorescent image to count positive reads32. The SlipChip has been described to perform digital PCR in a very simple and inexpensive format. Elongated wells in two plates are designed to overlap during sample load- ing. The fluidic path is broken up by slipping of the two plates that removes the overlap among wells and brings each well in contact with a reser- voir preloaded with oil to generate 1,280 nanolitre reaction compartments. After thermal cycling end- point fluorescence intensity is measured33. A microfluidic ‘megapixel’ digital PCR device was recently presented that uses surface tension-based sample partitioning and dehydration control to enable single molecule amplification in 1 million reactors of picolitre volume. The device has a dynamic range of 107, can detect a single- nucleotide-variant in one copy per 100,000 wild- type sequences and discriminates 1% difference in chromosome copy number34. Out of the forth- coming next generation dPCR platforms, closest to market are droplet techniques based on water- oil emulsions. The aqueous droplets act as micro- reactors in the bulk oil phase. Biochemical reac- tions in emulsion systems date back to 199835, and the first report on single molecule PCR in droplets came almost 10 years ago36. The early emulsion-based systems either had poor amplifica- tion efficiency or required a complicated work- flow. But major improvements during the last decade have led to the commercialisation of these platforms37. QuantaLife was first to launch with its Droplet Digital™ PCR system available at the end of last year38. The system is based on a droplet generator that partitions samples and mas- ter mix into nanolitre-sized droplets. DNA in the droplets is amplified by conventional thermal cycling whereafter a droplet reader (which is essentially a FACS) detects and measures the fluo- rescence of the droplets at a rate of 1,000 droplets per second. 32 samples can be analysed per hour. Raindance is expected to be next to launch39, ten- tatively early in 201240. Also Stokes Bio, which is part of Life Technologies, develops microfluidic solutions for quantitative PCR41.
Drug Discovery World Fall 2011
Future view
Digital PCR is here to stay and as awareness of the technology is increasing it will gain popularity. Commercialisation may be hampered by a com- plex IP landscape that may take some time to cross licence. In parallel, in-house development may be spurred by recent publication of ‘Idiot-proof emul- sion PCR’ protocol42. Routine diagnostic applica- tions that require IVD approval are still far ahead and will require the technology to mature. DDW
Dr Mikael Kubista is head of the department of gene expression at the institute of Biotechnology of the Czech Academy of Sciences in Prague43, and CEO and founder of the TATAA Biocenters (
www.tataa.com). TATAA Biocenters are leading providers of qPCR services in Europe and have an OpenArray from Life Technologies as well as a BioMark from Fluidigm. In collaboration with Life Technologies, TATAA offers digital PCR serv- ices in Europe. TATAA has an intensive R&D pro- gramme related to qPCR and has developed sever- al important products such as the dyes Chromofy and Visiblue, the 1-step extraction, RT, qPCR CelluLyser reagent, ValidPrime for RT-qPCR qual- ity control, and proprietary panels for the identifi- cation of optimum reference genes, for profiling of embryonic stem cells, and for profiling of circulat- ing tumor cells. TATAA also offers hands-on train- ing courses in qPCR and molecular diagnostics world-wide (
www.tataa.com/Courses/Courses. html) and arranges an annual qPCR symposium (
www.qpcrsymposium.eu).
Dr Anders Ståhlberg is working as Researcher at the Department of Pathology, Sahlgrenska Cancer Center, University of Gothenburg, Sweden. His primary research interest is to understand molecu- lar mechanisms in human sarcoma development and stem cell differentiation. He has developed sev- eral strategies for gene expression profiling, espe- cially at single cell level.
81
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92