SYBR® Green is a dye that intercalates into DNA and has many applications in molecular biology. One such application is real-time PCR, also known as qPCR. The intensity of the dye’s fluorescence increases with each successive PCR cycle and can be used to quantify DNA in the reaction. SYBR Green and other dye-based PCR methods are less expensive and more convenient than probe-based methods. There are disadvantages, as well. Nonspecific reaction products are more likely to develop with SYBR Green protocols, leading to increased background and false positives. Nonetheless, SYBR Green has a large and loyal following among researchers who are happy to work around the flaws of such a popular and time-tested technique.
The benefits of hot-start
Hot-start PCR methods help avert the problem of nonspecific amplification before the reaction begins. Hot-start technology is based on modified enzymes, primers or antibodies that inhibit the polymerase until denaturation temperature is achieved.
Eric Reyes, global product manager of qPCR reagents for Sigma Life Science, says its newest hot-start product, KiCqStart™ SYBR Green qPCR ReadyMix™, performs better than comparable products and is available in different formulations for the various real-time PCR instruments on lab benches today. The KiCqStart SYBR Green ReadyMix products are ready to use at 2X concentration with SYBR Green I, an antibody-mediated hot-start Taq DNA polymerase, nucleotides and MgCl2, plus buffers and stabilizers. Because it only needs primers and template, the often-tedious optimization for SYBR Green dye is expedited, and little to no optimization is actually required.
Some targets are more difficult to amplify than others. To address the problem of difficult targets, Life Technologies recently launched its SYBR Select Master Mix. The primary benefits of SYBR Select are a greater degree of specificity and reproducibility which, according to Sobha Pisharody, Life Technologies’ director of product management for genetic analysis, are best in class among SYBR mixes. SYBR Select contains a heat-labile uracil-DNA glycosylase (UDG) that removes contaminating templates that may interfere with amplification. “Having UDG in the mix can be problematic,” says Pisharody. “It sometimes chews up the reaction, because it's not always heat-killed.”
Advantages and Disadvantages of dUTPs
Amplification products sometimes linger and contaminate subsequent PCRs, resulting in false-positive results. One way of controlling that carryover is by replacing dTTP with dUTP in PCR products and then treating all subsequent starting reactions with UDG. UDG cleaves the uracil base from its phosphodiester backbone, but it does not affect natural DNA.
Heat-labile UDG cleans up contamination and then is itself deactivated upon heating. “It's easier for customers to be successful with their experiments,” Pisharody says. “One of the main problems that customers using SYBR have is the cycle of primer, test, redesign, test, redesign. The specificity of this mix enables that cycle to be shorter.”
However, the dUTP method does have drawbacks. It is only effective if all users of the same thermocycler are using a dUTP cleanup system. If even one member of the group is not, the control ceases to be effective. The use of dUTP has also been found inefficient because it reduces reaction efficiency, thereby increasing threshold cycle (Ct) values.
Bioline USA Inc., a Meridian Life Science Company, no longer sells PCR kits with dUTP for those reasons. Instead, it recommends an optimized protocol to allow high specificity combined with clean laboratory practices such as disposable consumables, filter tips and isolation of work areas for setup and amplification. Bioline's SensiFAST™ line of kits includes products for SYBR Green-based assays as well as probe-based assays.
Many laboratories are moving in the direction of fast protocols for qPCR, cutting, for example, a two-hour protocol down to 45 minutes or an hour. Although fast-protocol PCR is generally associated with an investment in expensive, fast-ramping instrumentation, it is also possible to achieve by tweaking parameters of a standard thermocycler, such as using a two-step protocol (combining annealing and extension) rather than the standard three-step. Reagents and consumables, such as newer hot-start polymerases, increase the time savings further.
Bio-Rad Laboratories' SsoAdvanced™ SYBR Green supermix is formulated with its Sso7d fusion enzyme. Sso7d is a dsDNA-binding protein that stabilizes the polymerase-template complex, increasing processivity, PCR inhibitor tolerance and reaction speeds. According to Bio-Rad, the supermix increases speed without affecting reaction specificity, sensitivity and efficiency.
Bio-Rad also offers the iTaq™ Universal SYBR Green Supermix. “The power of this SYBR Green supermix comes from universal instrument compatibility and ability to generate good qPCR data under broad reaction conditions,” says Ramesh Sathiyaa, PCR reagents product manager at Bio-Rad.
Thermo Scientific's DyNAmo™ Flash SYBR Green qPCR Kit is another product tailored for fast PCR; it is based on a hot-start version of modified Thermus brockianus DNA Polymerase, incorporating SYBR Green I. The T. brockianus polymerase gains stability from fusion of a nonspecific DNA binding domain and is inactive at room temperature. The ColorFlash version of the same kit incorporates a blue dye to help keep track of pipetting the master mix into reaction wells and a yellow dye for sample wells. It is easy to avoid pipetting errors, because when the sample and master mix are combined the resultant solution is green.
Alternatives to SYBR Green
Joe Donnehoffer, a technical service consultant for Roche Applied Science, tries to steer new customers away from SYBR Green: “If the customer is currently using SYBR Green and wanting to eliminate nonspecific products, we might encourage them to use our universal probe library.”
For those who strongly prefer SYBR Green-based qPCR, Donnehoffer recommends an alternative dye, the LightCycler 480 ResoLight Dye. That's because ResoLight, unlike SYBR Green, saturates molecule fragments, leading to an accurate melting curve. In contrast, the molecules in SYBR Green I do not saturate the DNA fragments and may “jump” from one strand to another during melting cycles.
Donnehoffer notes that loyalty to SYBR Green can be difficult to overcome. “Customers using SYBR Green … can be very reluctant to change to a different format.” For that reason, he does not expect SYBR Green to fade away any time soon. “It's not going anywhere.”
Life Technologies also offers an alternative dye, SYBR GreenER™, which is brighter and inhibits the PCR reaction less than SYBR Green I.
Primer design can make or break a qPCR experiment. Primer databases provide a source for tried-and-true primer designs. Some available databases include RTPrimerDB, PrimerBank and qPrimerDepot.
Predesigned primer sets are also available. For example, Qiagen's QuantiTect Primer Assays are kits designed for high sensitivity and specificity with 100% PCR efficiency for analysis of RNA. Qiagen offers assays for genes from human, rat, mouse, Drosophila, Arabidopsis and other species, with guaranteed results when used with its QuantiFast, QuantiTect, Rotor-Gene or FastLane SYBR Green kits. According to Qiagen, its QuantiFast SYBR Green kits offer time savings of up to 60%, even on standard thermocyclers.
OriGene is another vendor offering predesigned primers. Its SensiMix qPCR Primer Pairs are compatible with SYBR Green-based qPCR assays and make use of OriGene's proprietary algorithm, which is based on more than 10,000 qPCR experiments. The company’s collection covers the entire human and mouse genome.
Sigma has released predesigned KiCqStart Primers for the human, mouse and rat transcriptomes, says Tom Russell, global product manager of OEM, molecular diagnostics and qPCR probes. These assay primers are more economical than other predesigned primers on the market and are more readily available. “We have global manufacturing with facilities all over the world,” Russell says.
Although predesigned primers and assays are among the most premium options for assay optimization, in many cases the laboratory will earn the investment back in time saved that otherwise would have been spent in lengthy optimization protocols or in repetition of failed experiments and redesign and re-order of primers.
The image at the top of the page is from the Reproducibility data for Bio-Rad's SsoAdvanced SYBR Green Supermix.