Researchers at UC Irvine developed a method to amplify up to 100 different target sequences in a single DNA sample with Digital Polymerase Chain Reaction (dPCR) using multiple probes labeled by using combinatorial encoding of fluorescent color and/or intercalating dyes per reaction.
Polymerase chain reaction (PCR) is a technique used in nucleic acid detection in molecular biology. PCR works by amplifying a target sequence in a DNA sample in vitro, thereby generating millions of copies of a particular DNA sequence that can be purified and quantified after the reaction is completed. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.
An important improvement of PCR, is multiplex PCR, which allows for amplification of multiple target sequences in a single DNA sample, thereby saving time, effort and sample in the laboratory. Researches use quantitative PCR (qPCR) to perform multiplex PCR amplification (Multiplex qPCR), which provides more sensitive detection and quantification of the target sequence in real-time (rather than at completion). In particular, Multiplex qPCR uses fluorescent probes that bind and detect the target sequence. As the target sequence(s) amplifies using multiple primer sets, the fluorescent intensity of each increases, which can be quantified and visualized in real-time on a computer monitor. The predominant primer and enzyme/buffer system used is ThermoFisher’s TaqManTM.
Typically, as much as 5-plex qPCR is achievable in a PCR mixture by using fluorescently labeled TaqManTM probes, each one binding to a unique DNA sequence, which when amplified by TaqManTM polymerase, emit a brighter fluorescence signal at its specified spectral wavelength. The spectral frequency discrimination between different fluorophores, or reporter, attached to each probe sequence enables detection of five different target sequences, one for each fluorescent color that can be identified.
Multiplexing beyond 5-plex using Multiplex qPCR is difficult due to insufficient spectral wavelengths that can be optically distinguished using current state of the art fluorescence excitation and emission filter sets. In addition, no more than a single probe sequence can be used per spectral wavelength to further increase multiplexing ability, because one would be unable to determine what percentage of the total PCR amplified fluorescence intensity at that wavelength corresponds to more than a single probe sequence. For example, if two probes, A and B, were labeled with the same fluorescent marker, green, one wouldn't know if the amplified green signal was a composition of 100% probe A, 100% probe B, or some mixture of the two. For this same reason, multiplexing cannot be achieved using non-specific reporters such as intercalating dyes because one cannot differentiate amplified signal arising from any more than a single PCR primer pair. This is because these dyes report all double-stranded nucleotide strands with no distinction among sequence aside from the primer pairs that select for amplification. On the other hand, specific reporters such as target sequence specific DNA probes with quenchers are expensive, and increasing the number of different fluorescent reporters (e.g., for high-degree multiplexing) can quickly become cost-prohibitive.
As an alternative to qPCR, Digital PCR (dPCR) is a relatively new approach to nucleic acid detection and quantification, which works by partitioning a sample of DNA into multiple individual, parallel PCR reactions; some reactions contain the target sequence (positive) while others do not (negative). The dPCR reaction uses the TaqManTM system to amplify the target sequence million-fold or more and identify which reaction contains the target sequence using dye-labeled probes. The number of positive reactions represents the number or target sequences originally present in the DNA Sample. Thus, an absolute count of the number of target sequences is quantified using the fraction of negative reactions in a Poisson distribution.On-chip dPCR enables thousands of different reactions in parallel.
Using Digital PCR (dPCR), researchers at UC Irvine achieved 100-plex amplification with limited spectral bands using combinatorial color, temporal, and intensity encoding of probe sequences per reaction for a greater number of total signal readouts. Since each reaction contains a single target sequence, 100-percent of each PCR amplified fluorescence at a particular intensity signature corresponds a single probe sequence. Thus, using the method above, an absolute count of the number of up to 100 different target sequences originally in the DNA sample can be quantified. Moreover, less expensive non TaqMan-probe real-time PCR amplification indicators such as SYBR- or PicoGreen can be used to achieve multiplexed digital PCR based on temporal cues alone, intensity cues alone, or intensity and temporal cues combined, thus distinguishing primer pairs at greater degrees with significant cost reductions.
This invention can be used in medical and research laboratories engaged in genomics or molecular biology research. Additionally, the invention can be used to detect diseases, as well as medical, military, or environmental screening.
This invention yields much higher PCR multiplexing (100-plex) using dPCR, which translates to lower cost of research, less time spent in the lab, and less DNA sample used in any given reaction.
Utilizes a nanofluidic chip and associated read-out machines to run parallel reactions, which may prevent adoption of the technology.
|United States Of America||Published Application||20130178378||07/11/2013||2011-809|