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Advances In : Genomics - Amplifying Nucleic Acids
Some of today's life scientists would divide the history of biology into two parts: before and after the discovery of the polymerase chain reaction. This technique, which makes many copies of a section of DNA, spawned rapid advances in virtually every discipline related to chromosomes. Today, forensic scientists, high school students, basic researchers, and many others use this technique. Moreover, the experts interviewed here reveal some of the most recent advances in PCR.
by Mike May and Gary Heebner



ADVERTISERS

Leica Microsystems [Germany]
instruments and systems for imaging analysis, digital cameras
+49-6251-136-0,
http://www.confocal-microscopy.com
Leica Microsystems [USA] 847-405-0123

NanoDrop Technologies, Inc.
unique spectrophotometers based upon patented sample retention technology, allowing use of very small sample volumes
302-479-7707,
http://www.nanodrop.com

SANYO Sales & Marketing Corporation / SANYO Electric Biomedical Co., Ltd.
ultralow temperature freezers, CO2 incubators, pharmacy refrigerators, and autoclaves
http://www.sanyo-biomedical.co.jp

Takara Bio, Inc.
kits and reagents for molecular biology research, including genomics and proteomics
fax: +81 77-543-9254
http://www.takara-bio.co.jp/english

CONTENTS
PCR
Taq polymerase
Reverse transcription
Thermocycling
Quantitative PCR
Polymerase blends
PCR applications

This is the first of four supplements this year on genomics. The others will appear in the 10 June, 29 July, and 14 October issues of Science.

Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

In DNA: The Secret of Life, James D. Watson—codiscoverer of DNA's structure—and Andrew Berry wrote: ±You need large quantities of a given segment [of DNA], or gene, if you are going to sequence it." At first, scientists struggled to make copies of pieces of chromosomes. That all changed in 1983, when Kary Mullis discovered the polymerase chain reaction (PCR), and he later won a Nobel Prize for that work. Like the natural process of DNA replication, PCR depends on DNA polymerase. Watson and Berry called PCR ±a great leap forward." It generates large numbers of copies of any DNA fragment.

Over the past two decades, PCR changed many aspects of life science. Tuomas Tenkanen, research and development director for Finnzymes in Finland, says, "It is clear that PCR has been revolutionary to biological research." He adds, "It is amazing that such a simple method can speed up research so dramatically. PCR is now a basic tool, not only in biology labs but already in forensic and diagnostics labs." And many experts see even more PCR-related advances ahead. For instance, Nate Cosper, discovery and diagnostics consulting manager at Frost & Sullivan, which is a consulting firm that specializes in high technology and industrial markets, says that PCR ±has had a profound impact on basic research, clinical diagnostics, and environmental testing, and it is well poised to reach similar levels of importance in the detection of bioterrorism."

The Process of PCR
PCR starts with a template—the DNA that you want to replicate—and two primers. The primers are short chains of nucleotides that correspond to the nucleotide sequences on either side of the length of DNA of interest. These flanking sequences can be constructed in the laboratory or simply purchased from many biology suppliers, including Qiagen, Roche Applied Science, and Stratagene. The four nucleotides, DNA polymerase, buffers, and cofactors (such as magnesium) are then added. The conventional process takes several steps.

First, the mixture gets heated above 90 degrees Celsius to denature the target DNA, which makes it unwind into separate single strands. Once the strands separate, the primers bind to their complementary bases on the target DNA. Beginning at the primer, DNA polymerase reads the nucleic acid sequence and produces a complementary strand of DNA. That makes two newly assembled strands. Each time the process is repeated, which takes just a few minutes, the amount of DNA doubles. One of the workhorse enzymes in this process is Taq polymerase, which does the copying and was first isolated from the bacterium Thermus aquaticus.

The PCR process, though, continues to improve. According to Anne St. Louis, marketing director for gene expression at Stratagene, ±One of the most important recent advances is the ability to accurately amplify and clone from very small sample sizes, which is possible using proofreading enzymes like Pfu." She adds, "Being able to do that well with ultrahigh fidelity is key in order to avoid PCR-generated errors in early cycles, which would otherwise be amplified."

Making Complementary DNA
Although PCR works great with DNA, the process cannot directly amplify RNA, because RNA does not work as a template for DNA polymerase. To get PCR going on RNA, the RNA must first be converted to DNA with an enzyme from RNA retroviruses. These viruses enter a host cell, where they use reverse transcriptase to change their genetic material from RNA to DNA, which can be integrated into a host's genome. Once RNA is converted to DNA, it is called complementary DNA (cDNA), which is often created using messenger RNA as the template. The cDNA goes directly into conventional PCR. Various suppliers—including Ambion, BD Biosciences Clontech, and GE Healthcare—make reverse-transcriptase PCR kits.

When asked if amplifying RNA creates any special challenges, Gary Latham, senior scientist at Ambion, says, "Absolutely! RNA is inherently volatile and its levels fluctuate." That makes RNA more challenging to amplify. Actually, Latham says that the toughest part comes before amplification. He says, "You should ensure that the RNA is in an appropriate state to amplify and permit the proper data interpretations."

Also, there is more than one way to do reverse-transcriptase PCR. "You can put the reverse transcriptase and Taq in the same tube and run it from one reaction," Latham says, "or you can run it in two reactions and separately optimize the reverse transcription and PCR." But even more decisions must be made. Which primer will work best? There are three families of reverse-transcriptase enzymes to chose from, too. Some scientists even vary the reaction temperature for different genes.

To help with these complications, Ambion offers a variety of products. For example, Ambion's ArrayScript RT can make cDNA products as long as 9 kilobases. In addition, Ambion's TURBO DNAse removes any DNA that can contaminate the reverse transcription process. Latham says, "We can reduce DNA by several million fold." Then, Ambion's DNA-free removes the DNAse.

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Taking Care of the Temperature
The PCR process requires heating and cooling. In the past, scientists used several heating blocks or water baths set to different temperatures. That required attention to move the sample at just the right time. To eliminate that tedious process, manufacturers—including Applied Biosystems, Eppendorf, MJ Research, Roche Applied Science, Stratagene, and Techne—developed thermal cyclers, which consist of programmable heating and cooling devices. Now, a scientist can preset the temperature and the time periods for each cycle in advance and let the thermal cycler run unattended. According to Cosper, "The development of PCR technology, particularly the benchtop thermocycler, has been the single most important catalyst for the genomics revolution."

David Titus, marketing manager of amplification at Bio-Rad Laboratories, which owns MJ Research, says, "The original thermal cyclers were large, cumbersome to use, and inflexible in applications." He adds, "Bio-Rad's MJ cycler product line pioneered the use of Peltier thermoelectrics, which allowed a dramatic reduction in the size of thermal cyclers." This product line offers instruments with 10 different cycling block formats that include adjustable, heated lids. Titus says that these can be used for low-volume reactions, down to one microliter.

'several important advances in PCR performance have been made possible by a novel fusion enzyme technology—Sso7d—recently developed in Bio-Rad's MJ product group," says Titus. It is called iProof polymerase, and Titus says, "This enzyme provides the highest available accuracy—50-fold greater than Taq—while at the same time speeding up amplifications dramatically." He says that this enzyme can make a two-hour reaction run in 35 minutes for a 1-kilobase template.

Adding Quantification
When asked about one of the major advances in amplifying DNA, many experts mention the growing availability of quantitative PCR. Andy Felton, product manager for real-time PCR instruments at Applied Biosystems, which holds the patent on real-time PCR, says, "The major difference is that in regular PCR you measure the endpoint of the reaction process." To underscore the difference, he says, "After the 40 cycles of PCR, you measure the end result, and that is not quantitative." In real-time PCR, on the other hand, an investigator monitors the amplification as copies of DNA accumulate. Felton says, "You are going from a system with a vague answer about numbers to where you get specific, number-based answers."

Applied Biosystems offers a family of quantitative-PCR instruments. Felton calls the 7900HT ±a very high-end tool, often used for pharmaceutical research." Applied Biosystems also offers the 7500 and 7300 systems for real-time PCR. Felton says, "These systems provide a small footprint for small academic, hospital, or government labs. These also do the same range of applications." In addition, Applied Biosystems offers 400,000 gene expression assays for these systems. These newest platforms also reduce runtime from two hours to less than 40 minutes, and Applied Biosystems offers 1.8 million TaqMan SNP genotyping assays for real-time PCR systems.

As an example of using these instruments from Applied Biosystems, Greg Foltz, assistant professor of neurosurgery at the University of Iowa, analyzed glial blastomas—human brain tumors—with the Applied Biosystems Expression Array System and found a subset of genes implicated in the disease based on expression levels. Then, he used the 7900HT to pick out the genes that appeared to change in meaningful ways relative to the cancer.

Other companies also market quantitative PCR equipment. For instance, Roche Applied Science makes the LightCycler. While the LightCycler system can analyze all commercially available PCR probe chemistries, it specializes in hybridization probes. Hybridization probes bring together two different labels to allow fluorescence resonance energy transfer. This occurs when both probes bind, or hybridize, to the template DNA. Lance Brown, product manager at Roche Applied Science, explains: ±The fluorescent labeled probes let you analyze the PCR process while it occurs. You get to see what's happening in real time."

The current LightCycler 2.0 provides six fluorescent detection channels, which read light at 530, 560, 610, 640, 670, and 710 nanometers. As a result, Brown says, "The LightCycler 2.0 is very user-friendly to various dyes and different probe formats." He adds that this instrument plus its software ±can do about anything that you need to with PCR. You are only limited by the assay that you design."

Stratagene also offers three quantitative PCR instruments. According to Judy Macemon, Stratagene's director of marketing for instrumentation systems, "Our systems—the Mx4000, Mx3000P, and Mx3005P—support all fluorescent dyes and chemistries with customer-selected filters and the most powerful analysis software available." The Mx3000P, for example, includes four customer-selected filters. ±We find that scientists like the advantage of getting a top-performance, real-time instrument for their personal research use," says Macemon. The more recent Mx3005P includes five customer-selected filters, custom filter path selection for FRET chemistries, and probe/primer design software. The Mx4000—Stratagene's high-end quantitative PCR instrument—provided the most flexible options for experimental conditions and data analysis, plus customer-changeable filters.

Piles of Polymerases
Although all polymerases start making a copy at the 3-prime end of DNA, different ones provide various capabilities. St. Louis of Stratagene says, "Basic Taq is a really great workhorse." This polymerase is fast and robust for most any job that demands copying segments that are less than 1 kilobase long. However, when Taq incorporates an incorrect base, it tends to fall off the template. To solve that problem, says St. Louis, "Developers blend Taq with a proofreading enzyme so that errors can be corrected and amplification of longer sequences can be accomplished." A blend of Taq and a proofreader can copy segments greater than 10 kilobases in length.

In essence, different polymerase concoctions provide different speeds and fidelities. For example, Stratagene's PfuUltra is a polymerase that makes only one mistake every 2.5 million bases, on average. By comparison, Taq alone generates 1 mistake every 100,000 bases. St. Louis says, "There are numerous applications for high fidelity polymerases, such as cloning or site-directed mutagenesis to, say, change the sequence of a protein without creating additional unintended mutations."

Other companies—including Epicentre Technologies, Finnzymes, GE Healthcare, and USB Corporation—also aim at advancing the fidelity of polymerases. For example, the Phusion high-fidelity DNA polymerase from Finnzymes is a combination of proofreading DNA polymerase and a double-stranded DNA binding domain, which keeps the polymerase attached to the template. Tenkanen says, "The binding domain increases the processivity of the enzyme by 10 times over the enzyme alone." He adds, "What surprised us was that the fidelity increased after fusing the polymerase with the binding domain resulting in the highest fidelity DNA polymerase on the market."

Tenkanen says the Phusion polymerase product is especially useful for cloning. He says that they have one customer who does large amounts of PCR-based cloning and had always checked the sequence of the clones to make sure that they were correct. ±When they shifted to the Phusion product," says Tenkanen, "they found that they only got one mistake per 50 kilobases of sequenced PCR products. They realized that they could live with that error rate, and they stopped sequencing the clones."

Other companies try other modifications to PCR systems. In so-called hot start systems, for example, an inhibitory antibody binds the DNA polymerase until a high temperature releases the antibody and triggers the reaction. Hot start PCR systems are offered by Applied Biosystems, Invitrogen, Qiagen, and others.

Improvements in PCR can also arise by modifying components in the reaction mixtures, such as the buffer and magnesium. To keep it easier to get the mixtures right, companies—including Fermentas Life Sciences, Promega, Roche Applied Sciences, and Sigma-Aldrich—developed PCR systems that include all the needed reagents to perform this technique.

Outsourcing and Applications
In some cases, scientists or even companies prefer to send out PCR or cloning jobs to other companies, such as Evrogen, Imgenex, and Sigma-Genosys. According to Sergey A. Lukyanov, scientific director for Evrogen, "Modern science utilizes a number of techniques, some of which are time- and labor-consuming and require special equipment and special skills." He adds, "It is already impossible to set up all the necessary methods in one single lab."

Evrogen offers many services that utilize PCR amplification, isolation of differentially expressed genes by SSH (suppression subtractive hybridization), and cloning of full-length cDNA from different organisms by RACE (rapid amplification of cDNA ends). Lukyanov says, "cDNA normalization is another popular PCR-based technology invented by Evrogen and provided as a custom service." He adds, "Scientists use our normalized cDNA libraries for different purposes: high throughput cDNA sequencing aiming to describe the transcriptome of a new parasitic organism, creation of cDNA microarrays for expression profile analysis, functional screening for targets for the new medicines, etcetera."

PCR-based science also now appears in many applications. For instance, variations in genes can be used to develop drugs. For example, scientists at deCODE Genetics work in several clinical areas, from atherosclerosis and obesity to asthma and schizophrenia. Kari Stefansson, chief executive officer of deCODE, says, "We use microsatellites and SNPs, and we probably have more experience in using them in common diseases than anyone else." He adds, "Microsatellites are very good for genotyping, and you can do this with fewer microsatellites than SNPs." Currently, Stefansson's company has therapeutics under development from the drug discovery stage through Phase 2 trials.

Anyone in life science appreciates why Watson and Berry called PCR a great leap forward. As scientists apply this technique to an increasing number of problems, it appears that even more astounding uses of PCR lie ahead.

Mike May (mikemay1{at}verizon.net) is a freelance writer and editor based in Madison, Indiana, U.S.A. Gary Heebner
(gheebner{at}cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.
WEBLINKS
ADVERTISERS

Leica Microsystems [Germany]
instruments and systems for imaging analysis, digital cameras
+49-6251-136-0,
http://www.confocal-microscopy.com
Leica Microsystems [USA] 847-405-0123

NanoDrop Technologies, Inc.
unique spectrophotometers based upon patented sample retention technology, allowing use of very small sample volumes
302-479-7707,
http://www.nanodrop.com

SANYO Sales & Marketing Corporation / SANYO Electric Biomedical Co., Ltd.
ultralow temperature freezers, CO2 incubators, pharmacy refrigerators, and autoclaves
http://www.sanyo-biomedical.co.jp

Takara Bio, Inc.
kits and reagents for molecular biology research, including genomics and proteomics
fax: +81 77-543-9254
http://www.takara-bio.co.jp/english

FEATURED COMPANIES
and ORGANIZATIONS


Ambion, Inc.
RT-PCR kits and reagents
http://www.ambion.com

Applied Biosystems
thermal cyclers
http://www.appliedbiosystems.com

BD Biosciences Clontech
RT-PCR kits and reagents
http://www.clontech.com

Bio-Rad Laboratories
thermal cyclers
http://www.expressionproteomics.com

deCODE Genetics
biopharmaceutical company
http://www.decode.com

Epicentre Technologies
high fidelity Taq DNA polymerase
http://www.epicentre.com

Eppendorf AG
thermal cyclers
http://www.eppendorf.com

Evrogen
PCR services
http://www.evrogen.com

Fermentas Life Sciences
PCR master mixes
http://www.fermentas.com

Finnzymes
PCR kits and reagents
http://www.finnzymes.com

Frost & Sullivan
consulting/market research
http://www.frost.com

GE Healthcare
RT-PCR kits and reagents
http://www.amershambiosciences.com

IBC Life Sciences
scientific conference organizer
http://www.ibclifesciences.com/ScreenTech

Imgenex Corporation
PCR services,
http://www.imgenex.com

Invitrogen Corporation
hot start Taq DNA polymerase
http://www.invitrogen.com

MJ Research
thermal cyclers
http://www.mjr.com

Promega Corporation
PCR master mixes
http://www.promega.com

Qiagen GmbH
PCR kits and reagents
http://www.qiagen.com

Roche Applied Science
PCR kits and reagents
http://www.biochem.roche.com

Sigma-Aldrich Corporation
PCR master mixes
http://www.sigma-aldrich.com

Sigma-Genosys
PCR services
http://www.sigma-genosys.com

Stratagene
PCR kits and reagents
http://www.stratagene.com

Techne, Ltd.
thermal cyclers
http://www.techne.com

USB Corporation
high fidelity Taq DNA polymerase
http://www.usbweb.com

Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
This article was published
as a special advertising section
in the 11 February 2005 issue of Science


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