Explore the story of PCR, from the first bands visualized on a gel to the sophisticated molecular diagnostics practiced today…
History of PCR
The introduction of Polymerase Chain Reaction (PCR) in the 1980’s forever changed the field of molecular biology. A revolutionary technology, PCR is now a standard and indispensable technique for numerous medical and biological applications such as genome sequencing, evolutionary biology, forensic identification, and infectious disease detection. PCR is singularly responsible for the thriving industry of synthetic oligonucleotide manufacture.
Biosearch Technologies is proud to be part of PCR’s history. In 1982, a scientist named Kary Mullis at Cetus Corporation used Biosearch’s SAM I DNA synthesizer to create oligos for his first experiments, culminating in the invention of PCR. The basic process of PCR enabled manipulation of whole genes and accelerated monumental research such as the human genome project. PCR’s ability to identify and amplify enables characterization of all manner of life, whether the minute quantity of nucleic acid within single cell to a survey of genetic variation across an entire population. To learn more about the rise of the PCR, download the “30 Years of PCR” poster to retrace the historical footsteps of this seminal invention that forever changed molecular biology.
PCR captured the imagination of scientists who eagerly improved upon the method. The rigors of thermal cycling required a polymerase from Thermus aquaticus, which was isolated and expressed to provide an enzyme that remains active at elevated temperatures. In the 90’s the utility of Mullis’ invention was further enhanced with optical instrumentation and dye chemistry to measure DNA replication in real time. This method, termed real-time PCR, uses a fluorescent signal to correlate with the accumulation of product. Initially, fluorescent DNA binding dyes like ethidium bromide or SYBR Green were used to report the amplification. Introducing the dyes into the PCR reaction mixture before thermal cycling creates an elegant system to observe the reaction progress via a fluorescent readout. The kinetics of signal amplification are monitored to determine copy number, and this form of analysis is the basis for quantitative PCR (qPCR).
DNA binding dyes are combined with a primer-set for simple and economical assay development. This convenience comes at the expense of specificity since these dyes report signal from any spurious PCR product. Without additional scrutiny in the form of a melting curve analysis, off-target amplification can obscure the true result. The need for greater specificity motivated the development of fluorogenic probes—modified oligos pairing a fluorescent reporter to an acceptor dye. These probes are designed to hybridize to the target sequence between the primers, thus providing the assay with a third layer of specificity. Signal release requires target recognition, and so fluorogenic probes are ubiquitous in consequential assays requiring improved signal fidelity. They are critical components in molecular diagnostics where specificity is an absolute priority.
The functionality of fluorogenic probes are based on low background fluorescence when free in solution, accompanied by a large signal increase upon binding the target sequence. Signal-to-noise values are improved through careful selection of the dye partners.
Oligos with fluorescein (FAM) as a 5’ reporter and tetramethylrhodamine (TAMRA) as a 3' acceptor (FAM-TAMRA probes), were commonly synthesized prior to the availability of dark quenchers and still used by some groups today. When tethered at opposite ends of the same oligo, the dyes may interact in a process known as Förster Resonance Energy Transfer, or FRET, a quenching mechanism that transfers the excitation energy from the reporter to the acceptor according to their degree of spectral overlap, relative orientation, and position in space. Single-stranded oligos are thought to behave as a flexible coil to bring the dyes into close proximity for efficient quenching. Oligo hybridization forms a rigid duplex that increases the effective strand length, sufficient to disrupt the interaction between the dyes.
TAMRA works as an acceptor for some applications, but there are two drawbacks to this FRET setup: (1) TAMRA is also a fluorophore with emission that may contribute to background signal, and (2) A fluorescent acceptor limits multiplexing arrangements due to overlapping signal with other reporters in the reaction. Another acceptor dye called DABCYL is actually non-fluorescent, and so described as a “dark quencher.” However, DABCYL also has limited utility as a FRET quencher because it has an absorption maximum at only 453 nm, which is blue-shifted and far removed from the emission maxima of most fluorescent reporters. Dabcyl’s poor spectral overlap with common reporter dyes diminishes its utility for FRET quenching at longer wavelengths.
These limitations inspired the invention of the Black Hole Quencher® (BHQ®) dyes, a family of dark quenchers for incorporation into modified oligos. BHQ dyes represent the quintessential quencher in fluorogenic probes, where they partner with all common fluorescent reporters due to excellent spectral overlap. In addition to FRET quenching, they can also extinguish fluorescence through static quenching, a form of contact quenching where the reporter and quencher physically associate to form a ground state complex that is non-fluorescent. The versatile functionality of BHQ dyes is perfect for multiplexed assays that require quenching across the spectrum.
Learn more about the differences between contact quenching and FRET quenching mechanisms in BHQ probes for qPCR.
The excellent quenching efficiency of BHQ dyes make them the industry standard for qPCR, where they are incorporated into a variety of probe formats including Molecular Beacons, Scorpions® probes, and linear probes to name a few. They function according to different sequence conformations and reaction kinetics, but all involve a fluorescent reporter partnered with a dye acceptor. We highlight two probe types that are well suited for a diversity of applications.
BHQ Probes for qPCR
Traditional BHQ probes are linear probes typically 20 to 30 bases in length, incorporating a fluorophore at the 5’ end and a Black Hole Quencher (BHQ) dye at the 3' end. Signal is released upon hybridization of the probe to its complementary sequence, and made permanent when the 5' exonuclease activity of Taq polymerase cleaves off the fluorescent reporter. BHQ probes are ideal for detecting the presence of target sequences and quantifying their prevalence. In combination with reverse transcription, BHQ probes can be used to gauge the modulation of gene expression as an important measure of cellular activity during differentiation/development, in response to a drug treatment, or else environmental conditions. BHQ probes are also used for epidemiology and infectious disease detection: to combat outbreaks like the H1N1 influenza pandemic of 2009, and to screen for contaminating microbes that compromise food safety.
More information on BHQ probes can be found on the following webpage, along with an order form to request synthesis.
While traditional dual-labeled BHQ probes are perfectly suitable for many applications, certain demanding assays may require a more discerning probe type. For that purpose, BHQ probes can be further modified with a duplex stabilizing technology to achieve BHQplus® probes. This approach is often necessary to target difficult sequences such as AT-rich regions or single-nucleotide polymorphisms (SNPs). The unique chemistry of BHQplus probes permits the design of shorter sequences while maintaining the elevated melting temperatures required for qPCR. These compact probes are only 15-25 bases in length and so accommodated into a shorter sequence space. They provide increased specificity with respect to mismatches, as may be necessary for discrimination of closely related species. The BHQplus format is ideal when probes need improved specificity for binding the target sequence.
BHQ Probe Applications for Research and Industry
Whether academic research or a commercial process, scientists around the world use BHQ and BHQplus probes in their disciplines and industries. Probe-based qPCR is used for environmental screening, food testing, marker-assisted selection, pathogen detection, and many more. The versatile quenching of BHQ probes enables highly multiplexed assay design, while BHQplus probes provide an economical solution for SNP genotyping. Read on to learn how BHQ probes function in these qPCR applications.
Multiplexed real-time PCR combines several PCR assays into one reaction, to conserve sample material and minimize well-to-well variation. Multiple targets are amplified simultaneously but detected independently using fluorescent reporters with distinct spectra. The combined fluorescence is resolved into separate signal intensities that correlate with each amplification. Fluorescent reporters should be carefully chosen according to instrument optics and partnered with the appropriate Black Hole Quencher to achieve a robust multiplexed set.
Multiplexed PCR requires more time to implement compared to running the same assays independently, but the rewards justify the investment. A single multiplexed reaction yields additional data and confidence in the results. This approach achieves cost savings to an optimized assay that will be run repeatedly—ideal for commercial products or a high-throughput process.
View recommended dyes for multiplexing according to each real-time PCR instrument with our << Spectral Overlay Tool for Multiplexed qPCR >>
Probe-based SNP Genotyping
Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. A SNP is a single-base difference at a specific locus that varies among a population. SNPs are found throughout the genome and may contribute to disease if positioned within a functional element or other consequential region. Conversely, SNPs are also often used as markers for beneficial traits in the seed stocks of crops. SNPs are used to trace the evolution of a species and patterns of migration for evolutionary genomics. All of these investigations require probes with enhanced specificity to distinguish sequences according to a single mismatch.
Probe-based genotyping in combination with PCR uses the enhanced specificity of BHQplus probes to resolve the alleles. A common primer-set amplifies the target region, while two BHQplus probes compete to bind the sequence at the site of the SNP. Each labeled with a different fluorescent reporter, the probe that is fully complementary will dominate. The end-point fluorescence intensity of each probe is compared, with heterozygous and homozygous genotypes aligning to different regions of the scatter-plot.