PCR often gets taken for granted, but there are ways of making it faster, more accurate and easier to perform. Pete Moore investigates.
As a means of rapidly copying a selected template sequence from a DNA mixture in vitro, PCR by itself and in combination with other techniques has found a vast range of applications. These range from sequence detection and isolation for research, forensics and species identification to detecting mutations and polymorphisms and amplifying RNA-derived cDNAs for microarray analysis of gene expression. As well as standard PCR, the technique now comes in the form of real-time quantitative PCR (real-time PCR or qPCR). This uses fluorescent probes to monitor the amount of product at the end of each cycle, and real-time PCR machines look for the cycle at which they can first detect fluorescence. This relates to the number of copies of original template — the greater the number of starting copies, the fewer cycles are needed to reach fluorescence detection.
PCR can also be used to monitor RNA by adding a reverse transcriptase enzyme at the beginning to generate a DNA template. This reverse transcription PCR (RT-PCR) can then be taken a step further by adding the quantification protocols, resulting in real time RT-PCR.
There can be few life-science laboratories without a PCR thermal cycler tucked in a corner, happily churning out short DNA sequences to order with tried and tested protocols. But newer applications for PCR, such as single-nucleotide polymorphism (SNP) detection and screening, need faster throughput, and this is now achievable.
One approach is to abandon the traditional 96-well plate in favour of 384 wells or more. The new high-throughput 7900HT fast real-time PCR system from Applied Biosystems in Foster City, California, takes 96- and 384-well plates and runs a full set of amplification cycles in about 35 minutes. “Our new high-speed system can alleviate some of the burden of instrument sharing by reducing cycling time,” says Peter Dansky, senior director and general manager of core PCR at Applied Biosystems. If you need more than 384 wells, more manufacturers are now making 1,536-well plates, including Corning, of Corning, New York, evotec technologies of Hamburg, Germany, and KBiosciences of Hoddesdon, UK. The problem with the larger plates is getting robotic support, but Victor Crew, sales manager at KBiosciences, says that it is possible to modify automated pipettors like the Plate-Mate from Matrix Technologies in Beverly, Massachusetts, to use them. The Equator HTS and Latitude pipetting systems from Deerac Fluidics in Dublin, Ireland, will also take 1,536-well plates and will fill one in less than 15 seconds.
Throughput can also be increased by multiplexing — running more than one specific amplification reaction in a single tube. One approach is to use different colours of fluorigenic dyes to detect the different products. The new Mx3005P real-time PCR system from Stratagene of La Jolla, California, allows five different fluorigenic dyes to be used simultaneously. Using the company's FullVelocity probe-based real-time reagents, the machine will complete a 40-cycle two-step real-time PCR reaction in about 50 minutes. Cepheid of Sunnyville, California, make a real-time thermal cycler with four-colour optics and 96 independently programmable reaction holders that claims to get the job done in as little as 20 minutes.
There is probably a limit to how many dyes can be added to a single reaction tube. “Once you have got to four colours you have four sets of primers and four probes, and you have to stop these cross-reacting with each other and forming primer dimers,” says Chris Helps from the School of Clinical Veterinary Science at the University of Bristol. “When you increase the number of targets the reactions become very complex — you also need spectrally distinct dyes to minimize cross-talk between channels.”
Blowing hot and cold
How about really cutting the time down? The RapidCycler thermal cycler from Idaho Technology of Salt Lake City, Utah, blows blasts of hot and cold air through the reaction chamber, which gives near instantaneous temperature changes and rapid heat exchange with the samples. The RapidCycler 2 will do a 30-cycle run in 15 minutes, carrying 48 samples in either glass microcapillary tubes or the standard 1.5-mm reaction cuvettes that will also fit the widely used LightCycler, available from Roche Applied Science in Indianapolis, Indiana.
And it is possible to go faster still. The PCRJet thermocycler developed by a multidisciplinary team under the brand name MegaBase Research Products, in Lincoln, Nebraska, drives a mixture of hot and cold gas through the reaction chamber at 45 miles per hour. “The velocity of the air stream is so high that we are definitely in the turbulent region, which ensures that the heat transfer to the sample-containing capillary tubes is maximal. We can do 30 cycles of PCR with amplicons of anything from 100 to 600 base pairs in 2 to 3 minutes,” says Hendrik Viljoen of the department of chemical engineering at the University of Nebraska in Lincoln, one of the designers. “Going faster than 5 minutes doesn't really gain much for the working scientist, since it usually takes longer than that to mix the PCR reagents,” says team member Michael Nelson, “so we have backed off on speed and are now primarily concerned with system engineering for reliability and ease of use.”
The PCRJet takes eight samples of 20–100 ml at a time. “Talking to people in industry, we have found that in areas like infectious disease detection there is strong resistance to too small volumes. You need a big enough lump of sample because in the early stages of disease development there may be very few organisms present in a sample,” says Viljoen. PCRJet needs a fast enzyme, and it uses KOD Pol from Pyrococcus kodakaraensis from Toyobo Company of Osaka, Japan. Toyobo's Hideki Hayami, who is collaborating with MegaBase, says this can copy DNA at a rate of around 300 nucleotides per second.
Getting personal, Stratagene's Mx3000P is a four-colour optics real-time PCR machine for personal and small lab use, while the 46-well MJ Mini thermal cycler from Bio-Rad of Hercules, California (which recently acquired the manufacturers MJ Research) can be upgraded to a two-colour real-time machine with the retrofit of a MiniOpticon detection system.
Given that the physics of heating and cooling a PCR system can be problematic, a few pioneers are developing isothermal procedures — PCR at a uniform temperature — with an eye mainly on the clinical diagnostic market.
One approach is known as the ramification amplifying method (RAM). Invented by David Zhang of Mount Sinai School of Medicine in New York in 1994, RAM is licensed to Hamilton Thorne Biosciences of Beverly, Massachusetts, by Mount Sinai, which was recently granted an additional US patent on the method.
Target DNA is first isolated by capture probes linked to magnetic beads. A given target DNA is then detected by RAM, which employs a single-stranded DNA ‘C-probe’ that contains 3′ and 5′ sequences complementary to the target. If the C-probe hybridizes accurately with the target DNA, both ends of the probe bind close together and are joined by a ligase to form a circle. Once the circle is formed, this binds a primer at an internal site, which is extended by a strand-displacing DNA polymerase (for example, phi29 or Bst). As the polymerase travels around the circle it displaces the strand created on previous circuits, creating a long chain, which can itself can be duplicated by other primers and enzymes.
As the displaced DNAs are single stranded, primers can bind at a consistent temperature, removing the need for any thermocycling during amplification. However, “circle formation does require annealing of the C-probe to the targets and for long double-stranded DNA targets, a denaturation step would be beneficial,” explains David Lane, vice-president of research and development for Hamilton Thorne Biosciences.
Binding is highly specific, making it a useful tool for SNP detection. “Since RAM uses a universal primer, there is also no need for primer balancing, making high-level multiplexing as straightforward as adding multiple C-probes to an assay”, says Lane.
A completely different approach is that of Huimin Kong and colleagues, who came up with the idea of using a DNA helicase to separate the DNA strands rather than heat while working at New England Biolabs in Beverly, Massachusetts. “While other so-called isothermal techniques need an initial 95 °C DNA denaturation step, helicase-dependent amplification (HDA) can be performed in true isothermal conditions,” Kong says. Once the helicases have unwound the target, sequence-specific primers can bind to the single-stranded DNA and be extended, as in standard PCR.
Along with former colleagues, Kong has founded BioHelix Corporation in Beverly, with the aim of commercializing HDA. BioHelix's first commercial product is a teaching kit, marketed by the Carolina Biological Supply Company in Burlington, North Carolina, for students to carry out molecular diagnosis of sickle-cell anaemia in the classroom, without the need for an expensive thermocycler. The IsoAmp tHDA DNA amplification kit for research is due to launch this month.
Primers and probes made easy
For real-time PCR, the combinations of specific primers and the fluorescent oligonucleotide probes that detect template amplification are key. Researchers who hate designing probes and primers can now turn to online databases. PrimerBank, developed by Xiaowei Wang of the department of molecular biology at Harvard University and Massachusetts General Hospital, Boston, contains over 300,000 predicted primers for human and mouse genes generated computationally by a design algorithm. They have tested over 1,000 primer pairs and found a design success greater than 99% as defined by single PCR products and reasonable amplification efficiency, says Wang.
The Quantitative PCR Primer Database (QPPD), coordinated at the National Cancer Institute in Bethesda, Maryland, provides information about published primers and probes for quantitating human and mouse mRNA by RT-PCR.
Another source of published primer–probe sets for real-time PCR, with 3,376 entries so far, is RTprimerDB run by Jo Vandesompele and Filip Pattyn at the University of Ghent, Belgium, which can be searched by gene name, oligonucleotide sequence or NCBI's EntrezGene or SNP ID. “If people find the primer–probe pair they want they can click through to the PubMed identifier, and it also contains the details of the person who submitted the sequence,” says Vande-sompele. He hopes that coordinating the primer–probe sets that people use should increase standardization between labs, making it easier to compare results. Vandesompele also sees perils in the common use of a single housekeeper gene to normalize results in gene-expression studies, and his free geNorm applet for Excel will determine how many housekeeper genes you need for an accurate analysis. But he laments the fact that most analysis software only allows one housekeeper gene to be entered.
First described by Jesper Wengel in 1998, locked nucleic acids (LNA) are slowly making their way on to the PCR scene for use as probes. Their key advantage is their restricted conformational flexibility, which gives them great thermal stability and excellent mismatch discrimination when complexed with complementary DNA or RNA. At the Charité University Medical Centre in Berlin, Germany, Oliver Goldenberg and Lutz Hamann are using LNAs to quantify the species-specific 16S rDNA from multiple bacteria in a single PCR reaction. Their interest is in the intestinal flora that keep us healthy and they want a fast method of detecting the bacteria present without having to cultivate them. With a standard reporter dye such as SYBR Green, which fluoresces when it binds double-stranded DNA, only one specific feature can be assayed per sample — either a total bacterial count or identification of a single species. The widely used fluorescent resonance emission transfer (FRET)-based probes, such as the TaqMan system from Applied Biosystems of Foster City, California, aren't suitable either, as they require a recognition sequence of some 40 bases and “you will hardly find such long conserved regions in 16S rDNA,” says Goldenberg. “But the higher melting point of LNAs means that specificity can be achieved with shorter sequences.”
Designing LNA probes can pose problems (see ‘Simplifying the probe set’). “We have been testing LNA probes for SNP detection and have found the design parameters to be significantly different to standard TaqMan probes,” says Helps. He thinks the problem lies in the probe-design software: the most commonly used design software was written for the established fluorigenic probes but doesn't work as well for LNAs.
PCR has its twentieth birthday this year and has stood the test of time. Like the DNA it analyses it is evolving, and the next 20 years should be equally exciting.
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