An Introduction to qPCR
Polymerase chain reaction (PCR) is construed as one of the pioneering molecular biology techniques of the 90's. In fact, it was THE technique that completely revolutionized molecular biology. Its been more than a decade of PCR domination in molecular biology and clinical diagnostic labs, worldwide. The technique has evolved with these passing years, and now, it is quantitative PCR (qPCR) which is getting the same attention and is becoming one of the most widely used molecular biology techniques of this new millennium.
Quantitative PCR, as the name suggests, is a laboratory technique based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule in a given sample. The qPCR applications include gene quantification, pathogen detection and validation of drug targets. Quantitation is not a new idea in molecular biology. HIV viral loads in AIDS patients have been analyzed since decades now. These days, the focus is on quantitation because of the advancements in genomic research.
The basic real-time technique dates back to 1993, but it really took off in 1995-1996, when fluorescent techniques that monitor the accumulation of product, emerged. The theory of qPCR is not exactly like the practice. Theoretically, the number of templates double with each cycle of heating and cooling which results in exponential growth. To quantify the accumulated product, you need to know the the amount of starting material and how many reaction cycles were run. In practice though, the process is not as straight forward as it looks. After an exponential growth in the initial cycles, due to the limiting nature of reagents, a plateau phase eventually arrives and the reaction might not be exponential because of variations in reactions conditions or the accumulation of inhibitors.
Initial qPCR developments
Researchers at Roche Molecular Systems led the foundation of qPCR by developing a quantitative technique, based on the known property of Ethidium Bromide. When bound with DNA, it fluoresces upon excitation by Ultraviolet light. Later on, scientists discovered alternative superior methods for quantitation, other than the ones that involved Ethidium Bromide.
qPCR deploys three types of probe based approaches. The first approach uses Exonuclease probes which once bound to the target sequence are removed by taking advantage of the 3'-5' exonuclease activity of some DNA polymerases. One such proprietary probe category is of the TaqMan® probes, developed by Applied Biosystems. The earliest Exonuclease probes used P32, which was later on replaced in favor of fluorescent dyes.
The fluorescent dyes consist of quencher molecule and a reporter molecule. Separation of quencher from the donor increases the fluorescence. No cleavage occurs if the probe does not match the target sequence. While developing this technique, a multiplexing scenario came into picture for allelic discrimination. In qPCR assays for allele discrimination, two donor-quencher probes are used, one matches the gene sequence while the other matches the mutant sequence. Both these probes carry different fluorescent dyes, emitting fluorescent signal at different wavelength which distinguishes the target sequence.
The second probe based approach involved the development of new probes known as Hybridization probes. They increase the specificity and are used to discriminate between two alleles. A resonance energy transfer occurs when two hybridization probes which bind adjacent to each other and hybridize to an exact matched nucleotide target. If either of the probes encounters a mismatch, the resonance energy transfer does not occur and the fluorescence is much less intense. Thus, the fluorescence is directly proportional to hybridization between the probe and the target.
Apart from the two above mentioned techniques, other probe type chemistry includes Hairpin probes and Hairpin primers. They are named by the way they fold back on themselves. Hairpin probes, also known as Molecular Beacons, create a folded structure where both the ends are bound to each other while the loop binds to the target. The fluorescent reporter dye is present at one end and the quencher at the other. Molecular beacons can report the presence of specific nucleic acids from a homogeneous solution. In the presence of a complementary target, the "stem" portion of the beacon melts, resulting in the probe hybridizing to the target.
The major drawback of the above mentioned fluorescent probes is their costs. A comparatively inexpensive solution exists in form of SYBR® Green primers. SYBR® Green is the most widely used double-strand DNA-specific dye reported for qPCR. SYBR® Green binds to the minor groove of the DNA double helix. In the solution , the unbound dye exhibits little fluorescence. This fluorescence is substantially enhanced when the dye is bound to double stranded DNA. SYBR® Green remains stable under PCR conditions and the optical filter of the thermocycler can be affixed to harmonize the excitation and emission wavelengths.
Various research areas such as gene expression, allelic discrimination, biomarker discovery and drug discovery, frequently use qPCR as the basic diagnostic technique. Advancements in the standard qPCR protocols and instrumentation have led to highly sophisticated and accurate diagnostic tests and experiments being designed. These developments have been rammed by demands for increased throughput. lowering of costs, high assay sensitivity and reliable data normalization.
Challenges for the qPCR Chemistry
Despite these advancements in the qPCR technology, scientists constantly face hurdles in the following forms:
1)Sample contamination which results in false positives with the target DNA remaining unamplified.
2)Elaborate instrumentation control and sampling understanding is required hampering adoption of this technique.
3)High costs involved in setting up the system and carrying out the experiments makes it difficult to adopt in third world countries.