What's New With PCR

Let's start at the beginning. To do PCR, you need an enzyme, one able to withstand very high temperatures. Depending on your experiment, you might want one with high fidelity (decidedly not garden variety Taq), or you might need one that can read through secondary structure, a particular problem with RT-PCR given the complex structure of some RNAs. You might want one that can amplify long stretches, useful when working with genomic libraries.

Manufacturers and suppliers have literally searched the world over for enzymes with enhanced properties. And new enzymes and kits that improve on one or more property are continually being made. Here's a sampling of a few new ones that caught the editors' eyes.

Epicentre offers a blend of enzymes under the name FailSafe PCR System for both routine and challenging templates. The company claims that both long and difficult templates can be amplified consistently: PCR products greater than 20 kilobases can be obtained, and fragments with a GC content of greater than 85% have been amplified with FailSafe. The system blends a thermostable polymerase with a 3'–>5' proofreading activity to optimize fidelity and yield. The FailSafe system also works well for multiplexing, as the accompanying figure shows for the cystic fibrosis transmembrane conductance regulator gene (CFTR).

Multiplex PCR amplification of 5 CFTR exons

The FailSafe System comes with a set of 12 PreMixes, which provide dNPTs, buffer, various amounts of magnesium chloride, and FailSafe PCR enhancer (betaine), designed to increase consistency accuracy and sensitivity of PCR.

For more information: Epicentre Technologies, 1402 Emil St., Madison WI 53713. Tel: 800-284-8474.

MJ Research offers enzymes and kits from the Finnish company Finnzymes Oy. After testing almost 200 different thermophiles, the Finnish researchers selected one, Thermus brockianus, as the source of its enzymes. MJ sells these enzymes under the name DyNAzyme, and the latest one, DyNAzyme EXT, was designed to be to give strong bands without need for optimization, yet provide high fidelity equal to the best proofreading enzymes. DyNAzyme EXT is a mixture of DyNAzyme II DNA polymerase and a proofreading enzyme, and has applications in cloning PCR fragments, and amplifying long templates (up to 40 kb), and works well with difficult templates. The figure shows amplification of an "impossible" template, the upstream regulatory region of the chicken avidin gene, which had been refractory to PCR analysis.

Amplification of the chicken avidin gene. Lanes 1 through 6 were loaded with reactions using various Taq preparations, lane 5 DyNAzyme alone, lane 6, DyNAzyme with 5% DMSO.

For more information: MJ Research Inc., 590 Lincoln St., Waltham, MA 02451. Tel: 888-735-8437.

Promega offers native Pfu DNA Polymerase, isolated from geothermally heated sediments in Volcano, Italy, for applications requiring high fidelity. The enzyme, which sports a 3'–>5' exonuclease, has dramatically lower error rates compared to other thermostable enzymes, and according to company data, even other Pfu preparations. This, they say, is a function of the pH of the incubation buffer—the pH needs to be higher than 8.6 to get optimum fidelity.

Promega's Pfu can detect a sequence present in 10 copies (starting with 30 picograms of template) and for cloning, expressing, or mutagenesis experiments. Pfu DNA polymerase provides a low error rate.

For more information: Promega Corp., 2800 Woods Hollow Rd., Madison, WI 53711. Tel: 800-356-9526.

Roche Molecular Biochemicals offers a line of Expand enzyme blends that combine Taq with a proofreading polymerase in different ratios to optimize fidelity and yields for different fragment lengths. Expand Long Template PCR System, the preferred blend for most PCR applications, is optimized for two-fold greater yields and three-fold better fidelity when amplifying fragments of less than 5 kb. Two other polymerase blends, Expand Long Template and Expand 20kb^PLUS, deliver high fidelity and large yields for longer fragments.

For more information: Roche Molecular Biochemicals, 9115 Hague Rd., Indianapolis, IN 46250-0414. Tel: 800-428-5433.

Stratagene has put together a high fidelity DNA polymerase formulation that combines high yields, high fidelity, and amplifies broad target range of simple and complex targets. Its Herculase polymerase is a Pfu-based DNA polymerase formulation that works well at both ends of the spectrum—it amplifies extra-long genomic targets (up to 37 kilobases) as well as short amplicons (100–900 bases). DMSO can be added to amplifications to increase yield and extend length of difficult targets and those with high GC content. Since Pfu is the dominant polymerase, the results are high fidelity, greater, according to the company, than Taq-based blends and other proofreading enzymes.

Herculase successfully amplifies both very short and extremely long targets of varying complexity with high fidelity. Left: Long targets: Marker, 17kb, 30kb and 48kb targets. Right: Short targets: Marker, 105, 300, 500 bp targets.

For more information: Stratagene, 11011 North Torrey Pines Rd., La Jolla, CA 92037. Tel: 800-894-1304.

One-Step or Two-Step RT-PCR Kits
Epicentre's MasterAmp High Fidelity RT-PCR Kit offers accuracy and the option of either a one- or two-step protocol. This new high fidelity kit is ideal for cloning and/or expressing a RT-PCR product for applications that require minimal PCR error rates. The kit uses MMLV-RT Plus, which the company claims is the most reliable enzyme for full-length cDNA synthesis. It also includes MasterAmp TAQurate DNA Polymerase, giving the PCR step fidelity 3 times higher than Taq DNA polymerase.

MasterAmp High Fidelity is includes dNTPs, MgCl2, and buffer all in one vial, thus reducing the nuber of steps and the chances of contamination. This kit also contains both oligo (dT)18 and random nonamer primers. Finally, Epicentre's MasterAmp PCR Enhancement Technology improves the yield and specificity of amplification reactions, especially with templates having high G+C content or secondary structure.

Life Technologies offers a new kit for first-strand synthesis for RT-PCR called the GIBCO BRL Superscript First-Strand Synthesis System for RT-PCR. This kit has been optimized for sensitivity in two-step RT-PCR, and can provide detectable PCR products from as few as 50 to 100 starting RNA molecules, and from as little as 1 picogram of total RNA. In addition to high sensitivity, the company claims with this kit you'll get greater first-strand cDNA yields, and full-length synthesis up to 12 kb

Life Technologies has also made improvements to its one-step RT-PCR Kit. In the new SuperScript One-Step PCR System, the company has added Platinum Taq antibodies and a thermostable polymerase with proofreading capabilities. With this new system, PCR products can be obtained from < 1pg of total RNA for high-copy RNAs, and yield and length of product was improved. The addition of proofreading activity increased fidelity six-fold over Taq polymerase, and yielded targets as big as 8.9 kilobases.

For more information: Life Technologies Inc. (GIBCO BRL), 9800 Medical Center Dr., Rockville, MD 20849-6482. Tel: 800-828-6686.

MJ Research teams up DyNAzyme EXT with AMV-RT for its RT-PCR kit. AMV- reverse transcriptase has the advantage of being sufficiently heat-stable that first-strand synthesis of cDNA can be done at up to 60°C, melting out the RNA secondary structure that can interfere with RT-PCR. This results in longer reads than the other system (RNAse H minus MMLV reverse transcriptase) commonly used for RT-PCR. The inherent robustness of DyNAzyme EXT can generate a strong band from the cDNA, regardless of the target length or source.

Promega created the Access RT-PCR System for convenient single tube reactions. The kit combines AMV-RT with Tfl DNA Polymerase to provide detection sensitivity in the picogram range.

Promega's Reverse Transcription System provides both random hexamers and oligo dT primers in an AMV-RT system designed for two-step RT-PCR. The company states that the buffer system is compatible with their Taq DNA Polymerase for convenience and versatility. Both systems are now registered as a "General Purpose Reagent" for use in CLIA-governed molecular diagnostic laboratories.

QIAGEN Inc. offers a OneStep RT-PCR Kit, designed for fast and simple one-step RT-PCR. The new kit provides sensitive, high specificity one-step RT-PCR using any RNA. This kit blends two high-affinity reverse transcriptases to ensure highly efficient transcription of RNA amounts from 1 pg to 2 µg. The enzyme mix contains HotStarTaq DNA polymerase to improve specificity with hot-start PCR.

The specially formulated buffer provides robust conditions for both reverse transcription and PCR, ensuring high yields and high sensitivity. Q-solution, an innovative additive, is included in the kit to simplify RT-PCR with difficult templates. The kit is ideal for the most sensitive applications.

For more information: QIAGEN Inc., 28159 Ave. Stanford, Valencia, CA 91355. Tel: 800-426-8157.

For maximum RT-PCR fidelity and yield, Roche Molecular Biochemicals promotes its new C. therm Polymerase RT-PCR System in combination with either Expand High Fidelity (see above) or Taq polymerase for either one tube or two tube RT-PCR. Demonstrating optimal activity between 60°C and 70°C, this magnesium-dependent polymerase promises enhanced transcription efficiency at extremely high temperatures at which secondary structure degrades. The system produces one specific product—even in the presence of large amounts of non-specific RNA.

Stratagene offers two RT-PCR kits offering high fidelity. The ProSTAR HF single-tube RT-PCR system employs a one-step format combining MMLV reverse transcriptase and TaqPlus Precision DNA polymerase for high fidelity. cDNA synthesis and PCR amplification are performed in one tube with a single RT-PCR reaction buffer to provide convenience, speed and reduced risk of sample contamination.

The ProSTAR Ultra HF RT-PCR system provides a method for easy and efficient analysis of RNA in a two step method. This system contains all of the reagents for cDNA synthesis followed by high-fidelity PCR with PfuTurbo DNA polymerase. Stratagene believes its PfuTurbo DNA polymerase is the highest fidelity PCR enzyme available, generating highly accurate products with greater yields and longer length target capability.

Compiled by Laura DeFrancesco