qPCR
DNA diagnostics gets digitised
Quantitative real-time PCR (qPCR) has during the last two decades emerged as the preferred technology for nucleic acid analysis in routine as well as in research. qPCR has the sensitivity to detect a single molecule, the specificity to differentiate targets by a single nucleotide, and, because of its exponential nature, virtually unlimited dynamic range1.
W
ith the recent appearance of high throughput platforms represented by the OpenArray from Life
Technologies2, the BioMark from Fluidigm3, the LC1536 from Roche4 and the SmartChip from Wafergen5, can it get better? Well, classical qPCR is excellent platform technology and will dominate nucleic acid analyses years to come. But the expo- nential nature of qPCR, which is the key to its many advantages, is also limiting in some aspects. In particular, for example, multiplexing is chal- lenging. True multiplexing based on one tube amplification and separate detection of targets is limited by competition for reagents and is in prac- tice limited to two to four parallel reactions. In lab- oratories today most analyses performed are sin- gleplex. When multiple targets are assayed the sample is divided into aliquots that are analysed separately. If sample amount is limited the nucleic acid may be pre-amplified. There are several meth- ods to pre-amplify nucleic acids; some are linear while others are exponential, some are sequence specific while others are global, some target RNA while others target DNA. The methods are in gen- eral good, but they all require additional steps that add variation into the analysis, and they may also introduce bias. Another limitation of qPCR is pre- cision. Replicate qPCR amplification curves for high quality assays and with some 25 or more ini-
Drug Discovery World Fall 2011
tial template molecules appear usually very similar, and the standard deviation of the number of cycles required to reach threshold (Cq) is generally below 0.25 cycles and with some of the better instruments even below 0.1 cycle. From a technical perspective this reflects excellent instrument and assay per- formance (at a Cq of 25 cycles, SD of less than 0.25 cycles corresponds to a coefficient of varia- tion below 1%). However, variation in Cq reflects imprecision in the logarithm of the concentration; imprecision in concentration (regular linear scale) scale is substantially larger. This has impact on, for example, copy number determinations using qPCR. Precision in copy number determinations depends on the quality of the standard curve (ran- dom error among replicates, number of standards, dynamic range, and position on the standard curve), but is rarely within 50% (strictly, the confi- dence interval is symmetric in logarithmic scale). Hence, under advantageous conditions the preci- sion of qPCR is sufficient to detect a trisomy (dis- tinguishing between two and three copies) with acceptable false negative and positive rates, but not sufficient to measure smaller differences in copy number. Third limitation of qPCR is limited speci- ficity. True, a well-designed PCR assay amplifies with high preference a target with perfect sequence match for primers binding than sequences with even a single mismatch in a primer region, and
By Dr Mikael Kubista and Dr Anders Ståhlberg
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