Kary Mullis, Inventor of the PCR Technique, Dies

The Nobel laureate was a proponent of LSD, a consultant for O.J. Simpson’s legal defense, and the creator of a company that infused jewelry with celebrities’ DNA.

Aug 12, 2019

KERRY GRENS

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ABOVE: FLICKR, ERIK CHARLTON

Kary Mullis, whose invention of the polymerase chain reaction technique earned him the Nobel Prize in Chemistry in 1993, died of pneumonia on August 7, according to MyNewsLA.com. He was 74 years old.

According to a 1998 profile in The Washington Post, Mullis was known as a “weird” figure in science and “flamboyant” philanderer who evangelized the use of LSD, denied the evidence for both global warming and HIV as a cause of AIDS, consulted for O.J. Simpson’s legal defense, and formed a company that sold jewelry embedded with celebrities’ DNA. The opening paragraph of his Nobel autobiography includes a scene depicting a visit from Mullis’s dying grandfather in “non-substantial form.”

“He was personally and professionally one of the more iconic personalities science has ever witnessed,” Rich Robbins, the founder and CEO of Wareham Development, a real estate developer for a number of biotech companies, tells the Emeryville, California-based paper, the E’ville Eye.

See “PCR: Past, Present, & Future”

Mullis was born in North Carolina in 1944 and earned a chemistry degree from Georgia Tech and a PhD in biochemistry from the University of California, Berkeley. In the early 1980s, when Mullis was working for the biotech company Cetus Corp in Emeryville, he developed the DNA replication technique polymerase chain reaction (PCR)—one of the most widely used methods in molecular biology.

Writing in The Scientist in 2003, Mullis described his first attempt at PCR in 1983 as “a long-shot experiment. . . . so [at midnight] I poured myself a cold Becks into a prechilled 500 ml beaker from the isotope freezer for luck, and went home. I ran a gel the next afternoon [and] stained it with ethidium. It took several months to arrive at conditions [that] would produce a convincing result.”

Both Science and Nature rejected the resulting manuscript, which was ultimately published in Methods in Enzymology in 1987.

By then, Mullis had left Cetus went on to consult for a number of biotech firms, including what the San Francisco Chronicle calls “harebrained business ventures,” as well as for big names in the life sciences, such as Abbott Labs and Eastman Kodak. In the mid-2000s, he formed a company called Altermune with the goal of developing a therapeutic to redeploy a person’s existing antibodies against new pathogens.

Mullis is survived by his wife Nancy, three children, and two grandchildren.

Kerry Grens is a senior editor and the news director of The Scientist. Email her at kgrens@the-scientist.com.

Keywords:

kary mullis

nutshell

obituaries

obituary

PCR

techniques

https://www.the-scientist.com/news-opinion/kary-mullis--inventor-of-the-pcr-technique--dies-66256

Polymerase chain reaction

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"PCR" redirects here. For other uses, see PCR (disambiguation).

A strip of eight PCR tubes, each containing a 100 μl reaction mixture

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Developed in 1983 by Kary Mullis,^[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.^[2][3] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.^[4]
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Placing a strip of eight PCR tubes, each containing a 100 μl reaction mixture, into the PCR machine

Contents 1 PCR principles and procedure 1.1 Procedure 2 PCR stages 2.1 PCR optimization 3 Application of PCR 3.1 Selective DNA isolation 3.2 Amplification and quantification of DNA 3.3 PCR in diagnosis of diseases 4 Variations on the basic PCR technique 5 History 5.1 Patent wars 6 References 7 External links

PCR principles and procedure

Figure 1a: A thermal cycler for PCR

Figure 1b: An older model three-temperature thermal cycler for PCR

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.^[5]
A basic PCR set up requires several components and reagents.^[6] These components include:

• DNA template that contains the DNA region (target) to be amplified.
• Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
• Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
• Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
• Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
• Divalent cations, magnesium or manganese ions; generally Mg²⁺ is used, but Mn²⁺ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn²⁺ concentration increases the error rate during DNA synthesis^[7]
• Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.

Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.^[8]

• Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.^[9]

• Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

• Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

• Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,^[10][11] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

• Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

• Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).

 PCR stages

The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.^[12]
Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

 PCR optimization

Polymerase chain reaction optimization

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The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization have been developed by molecular biologists to improve PCR performance and minimize failure.

Contents 1 Contamination and PCR 2 Hairpins 3 Polymerase errors 4 Magnesium concentration 5 Size and other limitations 6 Non-specific priming 7 Primer dimers 8 Deoxynucleotides 9 References

Contamination and PCR

The PCR method is extremely sensitive, requiring only a few DNA molecules in a single reaction for amplification across several orders of magnitude. Therefore, adequate measures to avoid contamination from any DNA present in the lab environment (bacteria, viruses, or human sources) are required. Because products from previous PCR amplifications are a common source of contamination, many molecular biology labs have implemented procedures that involve dividing the lab into separate areas.^[1] One lab area is dedicated to preparation and handling of pre-PCR reagents and the setup of the PCR reaction, and another area to post-PCR processing, such as gel electrophoresis or PCR product purification. For the setup of PCR reactions, many standard operating procedures involve using pipettes with filter tips and wearing fresh laboratory gloves, and in some cases a laminar flow cabinet with UV lamp as a work station (to destroy any extraneomultimer formation). It is routinely assessed with a (negative) control PCR reaction. This control reaction is set up in the same way as the experimental PCRs, but without template DNA added, and is performed alongside the experimental PCRs.

 Hairpins

Secondary structures in the DNA can result in folding or knotting of DNA template or primers, leading to decreased product yield or failure of the reaction. Hairpins, which consist of internal folds caused by base-pairing between nucleotides in inverted repeats within single-stranded DNA, are common secondary structures and may result in failed PCRs.
Typically, primer design that includes a check for potential secondary structures in the primers, or addition of DMSO or glycerol to the PCR to minimize secondary structures in the DNA template^{[citation needed]}, are used in the optimization of PCRs that have a history of failure due to suspected DNA hairpins. http://www.promega.com/pnotes/65/6921_27/6921_27_core.pdf

 Polymerase errors

Taq polymerase lacks a 3' to 5' exonuclease activity. Thus, Taq has no error-proof-reading activity, which consists of excision of any newly misincorporated nucleotide base from the nascent (=extending) DNA strand that does not match with its opposite base in the complementary DNA strand. The lack in 3' to 5' proofreading of the Taq enzyme results in a high error rate (mutations per nucleotide per cycle) of approximately 1 in 10,000 bases, which affects the fidelity of the PCR, especially if errors occur early in the PCR with low amounts of starting material, causing accumulation of a large proportion of amplified DNA with incorrect sequence in the final product.^[2]
Several "high-fidelity" DNA polymerases, having engineered 3' to 5' exonuclease activity, have become available that permit more accurate amplification for use in PCRs for sequencing or cloning of products. Examples of polymerases with 3' to 5' exonuclease activity include: KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis KOD1; Vent, which is extracted from Thermococcus litoralis; Pfu DNA polymerase, which is extracted from Pyrococcus furiosus; and Pwo, which is extracted from Pyrococcus woesii.

Magnesium concentration

Magnesium is required as a co-factor for thermostable DNA polymerase. Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction.^[3] Some of the components of the reaction mixture such as template concentration, dNTPs and the presence of chelating agents (EDTA) or proteins can reduce the amount of free magnesium present thus reducing the activity of the enzyme.^[4] Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations and so results in decreased specificity of the reaction. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.^[3][4] Inadequate thawing of MgCl₂ may result in the formation of concentration gradients within the magnesium chloride solution supplied with the DNA polymerase and also contribute to many failed experiments.^[4]

 Size and other limitations

PCR works readily with a DNA template of up to two to three thousand base pairs in length. However, above this size, product yields often decrease, as with increasing length stochastic effects such as premature termination by the polymerase begin to affect the efficiency of the PCR. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These are polymerases fused to a processivity-enhancing DNA-binding protein, enhancing adherence of the polymerase to the DNA.^[5][6]
Other valuable properties of the chimeric polymerases TopoTaq and PfuC2 include enhanced thermostability, specificity and resistance to contaminants and inhibitors.^[7][8] They were engineered using the unique helix-hairpin-helix (HhH) DNA binding domains of topoisomerase V^[9] from hyperthermophile Methanopyrus kandleri. Chimeric polymerases overcome many limitations of native enzymes and are used in direct PCR amplification from cell cultures and even food samples, thus by-passing laborious DNA isolation steps. A robust strand-displacement activity of the hybrid TopoTaq polymerase helps solving PCR problems with hairpins and G-loaded double helices, because helices with a high G-C context possess a higher melting temperature.^[10]

 Non-specific priming

Non-specific binding of primers frequently occurs and can be due to repeat sequences in the DNA template, non-specific binding between primer and template, and incomplete primer binding, leaving the 5' end of the primer unattached to the template. Non-specific binding is also often increased when degenerate primers are used in the PCR. Manipulation of annealing temperature and magnesium ion (which stabilise DNA and RNA interactions) concentrations can increase specificity. Non-specific priming during reaction preparation at lower temperatures can be prevented by using "hot-start" polymerase enzymes whose active site is blocked by an antibody or chemical that only dislodges once the reaction is heated to 95˚C during the denaturation step of the first cycle.
Other methods to increase specificity include Nested PCR and Touchdown PCR.
Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.^[11]
Touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers will avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will "swamp out" any specific sequences because of the exponential nature of polymerase amplification.
The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles (the number of individual cycles and increments of temperature decrease is chosen by the experimenter). The primer will anneal at the highest temperature which is least-permissive of nonspecific binding that it is able to tolerate. Thus, the first sequence amplified is the one between the regions of greatest primer specificity; it is most likely that this is the sequence of interest. These fragments will be further amplified during subsequent rounds at lower temperatures, and will out compete the nonspecific sequences to which the primers may bind at those lower temperatures. If the primer initially (during the higher-temperature phases) binds to the sequence of interest, subsequent rounds of polymerase chain reaction can be performed upon the product to further amplify those fragments.

 Primer dimers

Annealing of the 3' end of one primer to itself or the second primer may cause primer extension, resulting in the formation of so-called primer dimers, visible as low-molecular-weight bands on PCR gels.^[12] Primer dimer formation often competes with formation of the DNA fragment of interest, and may be avoided using primers that are designed such that they lack complementarity—especially at the 3' ends—to itself or the other primer used in the reaction. If primer design is constrained by other factors and if primer-dimers do occur, methods to limit their formation may include optimisation of the MgCl₂ concentration or increasing the annealing temperature in the PCR.^[12]

Deoxynucleotides

Deoxynucleotides (dNTPs) may bind Mg²⁺ ions and thus affect the concentration of free magnesium ions in the reaction. In addition, excessive amounts of dNTPs can increase the error rate of DNA polymerase and even inhibit the reaction.^[3][4] An imbalance in the proportion of the four dNTPs can result in misincorporation into the newly formed DNA strand and contribute to a decrease in the fidelity of DNA polymerase.^[13]

References

1. ^ Balin BJ, Gérard HC, Arking EJ, et al. (1998). "Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain". Med. Microbiol. Immunol. 187 (1): 23–42. doi:10.1007/s004300050071. PMID 9749980. 
2. ^ Eckert KA, Kunkel TA (August 1991). "DNA polymerase fidelity and the polymerase chain reaction". PCR Methods Appl. 1 (1): 17–24. PMID 1842916. http://www.genome.org/cgi/pmidlookup?view=long&pmid=1842916. 
3. ^ ^{a b c} Markoulatos P, Siafakas N, Moncany M (2002). "Multiplex polymerase chain reaction: a practical approach". J. Clin. Lab. Anal. 16 (1): 47–51. doi:10.1002/jcla.2058. PMID 11835531. 
4. ^ ^{a b c d} Nucleic acid amplification protocols and guidelines. http://www.promega.com/paguide/chap1.htm. 
5. ^ Pavlov AR, Belova GI, Kozyavkin SA, Slesarev AI (2002). "Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases". Proc Natl Acad Sci. 99 (21): 3510–13515. doi:10.1073/pnas.202127199. . PMID 12368475. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=129704. 
6. ^ Demidov VV (2002). "A happy marriage: advancing DNA polymerases with DNA topoisomerase supplements". Trends Biotechnol. 20 (12): 491. doi:10.1016/S0167-7799(02)02101-7. 
7. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends Biotechnol. 22 (5): 253–260. doi:10.1016/j.tibtech.2004.02.011. PMID 15109812. 
8. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Thermostable Chimeric DNA Polymerases with High Resistance to Inhibitors". DNA Amplification: Current Technologies and Applications. Horizon Bioscience. pp. 3–20. ISBN 0-9545232-9-6. http://www.horizonpress.com/hsp/abs/absdna.html. 
9. ^ Forterre P (2006). "DNA topoisomerase V: a new fold of mysterious origin". Trends Biotechnol. 24 (6): 245–247. doi:10.1016/j.tibtech.2006.04.006. PMID 16650908. 
10. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes". In Kieleczawa J. DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett. pp. 241–257. ISBN 0-7637338-3-0. http://bioscience.jbpub.com/catalog/0763733830/table_of_contents.htm. 
11. ^ "Electronic PCR". NCBI - National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/sutils/e-pcr/. Retrieved 13 March 2012. 
12. ^ ^{a b} Kramer MF, Coen DM (August 2006). "Enzymatic amplification of DNA by PCR: standard procedures and optimization". Curr Protoc Cytom Appendix 3: Appendix 3K. doi:10.1002/0471142956.cya03ks37. PMID 18770830. 
13. ^ Kunz BA, Kohalmi SE (1991). "Modulation of mutagenesis by deoxyribonucleotide levels". Annu. Rev. Genet. 25: 339–59. doi:10.1146/annurev.ge.25.120191.002011. PMID 1812810.
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.^[13][14] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.^[6] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as formamide, in buffer systems may increase the specificity and yield of PCR.^[15] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.^[16]

Application of PCR

Applications of PCR

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This article is an orphan, as few or no other articles link to it. Please introduce links to this page from related articles; suggestions may be available. (May 2008)

This page assumes familiarity with the terms and components used in the Polymerase Chain Reaction (PCR) process.

The Polymerase Chain Reaction (PCR) has found widespread application in many areas of genetic analysis. This is a list of some of these applications:

Contents  [hide]  1 Medical applications 2 Infectious disease applications 3 Forensic applications 4 Research applications 5 Others 6 References

[edit] Medical applications

PCR has been applied to a large number of medical procedures:

• The first application of PCR^[1] was for genetic testing, where a sample of DNA is analyzed for the presence of genetic disease mutations. Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease. DNA samples for Prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis is also essential to Preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations.

• PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. As of 2008, there is even a proposal to replace the traditional antibody-based tests for blood type with PCR-based tests.^[2]

• Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient.

[edit] Infectious disease applications

Characterization and detection of infectious disease organisms have been revolutionized by PCR:

• The Human Immunodeficiency Virus (or HIV), responsible for AIDS, is a difficult target to find and eradicate. The earliest tests for infection relied on the presence of antibodies to the virus circulating in the bloodstream. However, antibodies don't appear until many weeks after infection, maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the infection don't affect the antibodies. PCR tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells.^[3] Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified.

• Some disease organisms, such as that for Tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy. The effects of therapy can also be immediately evaluated.

• The spread of a disease organism through populations of domestic or wild animals can be monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be detected and monitored. The sub-types of an organism that were responsible for earlier epidemics can also be determined by PCR analysis.

[edit] Forensic applications

The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics:

• In its most discriminating form, Genetic fingerprinting can uniquely discriminate any one person from the entire population of the world. Minute samples of DNA can be isolated from a crime scene, and compared to that from suspects, or from a DNA database of earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation. Evidence from decades-old crimes can be tested, confirming or exonerating the people originally convicted.

• Less discriminating forms of DNA fingerprinting can help in Parental testing, where an individual is matched with their close relatives. DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual biological father of a newborn can also be confirmed (or ruled out).

[edit] Research applications

PCR has been applied to many areas of research in molecular genetics:

• PCR allows rapid production of short pieces of DNA, even when nothing more than the sequence of the two primers is known. This ability of PCR augments many methods, such as generating hybridization probes for Southern or northern blot hybridization. PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, enabling analysis even from very small amounts of starting material.

• The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily be produced from a patient with a genetic disease mutation. Modifications to the amplification technique can extract segments from a completely unknown genome, or can generate just a single strand of an area of interest.

• PCR has numerous applications to the more traditional process of DNA cloning. It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities. Using a single set of 'vector primers', it can also analyze or extract fragments that have already been inserted into vectors. Some alterations to the PCR protocol can generate mutations (general or site-directed) of an inserted fragment.

• Sequence-tagged sites is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone. The Human Genome Project found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories.

• An exciting application of PCR is the phylogenic analysis of DNA from ancient sources, such as that found in the recovered bones of Neanderthals, or from frozen tissues of Mammoths. In some cases the highly degraded DNA from these sources might be reassembled during the early stages of amplification.

• A common application of PCR is the study of patterns of gene expression. Tissues (or even individual cells) can be analyzed at different stages to see which genes have become active, or which have been switched off. This application can also use Q-PCR to quantitate the actual levels of expression

• The ability of PCR to simultaneously amplify several loci from individual sperm^[4] has greatly enhanced the more traditional task of genetic mapping by studying chromosomal crossovers after meiosis. Rare crossover events between very close loci have been directly observed by analyzing thousands of individual sperms. Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all without having to wait (or pay for) the long and laborious processes of fertilization, embryogenesis, etc.

 Others

PCR is also important in answering basic scientific questions. In the field of evolutionary biology, PCR has been used to establish relationships among species. In anthropology, it has been used to understand ancient human migration patterns. In archaeology, it has been used to help identify ancient human remains. Paleontologists have used PCR to amplify DNA from extinct insects preserved in amber for 20 million years. The Human Genome Project, which had a goal of determining the sequence of the 3 billion base pairs in the human genome, relied heavily on PCR. The genes responsible for a variety of human diseases have been identified using PCR. For example, a PCR technique called multiplex PCR identifies a mutation in a gene in boys suffering from Duchenne muscular dystrophy. PCR can also be used to search for DNA from foreign organisms such as viruses or bacteria.

 References

1. ^ Saiki RK et al. "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia" Science vol. 230 pp. 1350-54 (1985).
2. ^ Quill E "Blood-Matching Goes Genetic" Science Magazine (14 March 2008) pp. 1478-1479.
3. ^ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
4. ^ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989).

Selective DNA isolation

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.^{[citation needed]}
Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E. coli) can be rapidly screened by PCR for correct DNA vector constructs.^[17] PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.^{[citation needed]}
Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms.^{[citation needed]}

Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

 Amplification and quantification of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar.^[18]
Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

• See also Use of DNA in forensic entomology

PCR in diagnosis of diseases

PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest-developed in cancer research and is already being used routinely. (See the studies cited in the EUTOS For CML study article at http://www.eutos.org/content/molecular_monitoring/information/pcr_testing/, especially notes 10-13.) PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000-fold higher than that of other methods.^{[citation needed]}
PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.^[19]
Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).

 Variations on the basic PCR technique

Main article: Variants of PCR

• Allele-specific PCR: a diagnostic or cloning technique based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.^[20] See SNP genotyping for more information.

• Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.^[21]

• Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.^[22] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.^[23]

• Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.^[24]

• Hot start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95°C) before adding the polymerase.^[25] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody^[9][26] or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

• Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.^[27]

• Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.^[28]

• Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.^[29]

• Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,^[30] and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

• Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.^[31]

• Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).

• Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis.

• Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

• Overlap-extension PCR or Splicing by overlap extension (SOE) : a genetic engineering technique that is used to splice together two or more DNA fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations; the technique enables creation of specific and long DNA constructs.

• Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR (or QF-PCR) methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.

• Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).

• Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR^[32] (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR^[33] (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).

• Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.^[34]

• Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the T_m of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer T_m. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.^[35]

• PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.^[36][37]

• Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer — which can lead to artefactual 'noise')^[38] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),^[39] 5'RACE LaNe^[40] and 3'RACE LaNe.^[41]

• In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR) refers to computational tools used to calculate theoretical polymerase chain reaction results using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome.

History

A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro.^[42] However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.^[43]
When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car.^[44] He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."^[45] He was awarded the Nobel Prize in Chemistry in 1993 for his invention,^[4] seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle (see below).
At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.^[2] So the early procedures for DNA replication were very inefficient and time consuming, and required large amounts of DNA polymerase and continuous handling throughout the process.
The discovery in 1976 of Taq polymerase — a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments^[10] such as hot springs — paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,^[11] thus obviating the need to add new DNA polymerase after each cycle.^[3] This allowed an automated thermocycler-based process for DNA amplification.

Kary Mullis

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Kary Mullis
Born	(1944-12-28) December 28, 1944 (age 67)Lenoir, North Carolina, United States
Fields	Molecular biology
Alma mater	Georgia Institute of Technology University of California, Berkeley, PhD
Known for	Invention of polymerase chain reaction
Notable awards	Nobel Prize in Chemistry and Japan Prize (both in 1993)

Kary Banks Mullis (born December 28, 1944) is a Nobel Prize winning American biochemist, author, and lecturer. In recognition of his improvement of the polymerase chain reaction (PCR) technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith^[1] and earned the Japan Prize in the same year. The process was first described by Kjell Kleppe and 1968 Nobel laureate H. Gobind Khorana, and allows the amplification of specific DNA sequences.^[2][3] The improvements made by Mullis allowed PCR to become a central technique in biochemistry and molecular biology, described by The New York Times as "highly original and significant, virtually dividing biology into the two epochs of before P.C.R. and after P.C.R."^[4]
Since winning the Nobel Prize, Mullis has been criticized in The New York Times for promoting ideas in areas in which he has no expertise.^[5] He has promoted AIDS denialism,^{[6][7][8][9][10][11]} climate change denial^[6] and his belief in astrology.^[5][6]

Contents  [hide]  1 Early life and education 2 Career 2.1 PCR and other inventions 2.2 Accreditation of the PCR technique 3 Personal views 4 Use of LSD 5 Extraterrestrial life 6 Personal life 7 Books authored 7.1 Awards and honors 8 See also 9 References 10 Further reading 11 External links 11.1 Interviews

[edit] Early life and education

Mullis was born in Lenoir, North Carolina, near the Blue Ridge Mountains,^[12] on December 28, 1944. His family had a background in farming in this rural area. As a child, Mullis recalls, he was interested in observing organisms in the countryside.^[3] He grew up in Columbia, South Carolina,^[3] where he attended Dreher High School.
Mullis earned a Bachelor of Science degree in chemistry^[12] from the Georgia Institute of Technology in Atlanta in 1966, during which time he got married and started a business.^[13] He then received a Ph.D. in biochemistry from the University of California, Berkeley in 1972; his research focused on synthesis and structure of proteins.^[3] Following his graduation, Mullis became a postdoctoral fellow in pediatric cardiology at the University of Kansas Medical School, going on to complete two years of postdoctoral work in pharmaceutical chemistry at the University of California, San Francisco.

[edit] Career

After receiving his PhD, Mullis left science to write fiction, but quit and became a biochemist at a medical school in Kansas City.^[13] He then managed a bakery for two years.^[4] Mullis returned to science at the encouragement of friend Thomas White, who later got Mullis a job with the biotechnology company Cetus Corporation of Emeryville, California.^[3][4] Mullis worked as a DNA chemist at Cetus for seven years; it was there, in 1983, that Mullis invented his prize-winning improvements to the polymerase chain reaction.^[14] After leaving Cetus in 1986, Mullis served as director of molecular biology for Xytronyx, Inc. in San Diego for two years. Mullis has consulted on nucleic acid chemistry for multiple corporations.^[4]
In 1992, Mullis founded a business with the intent to sell pieces of jewelry containing the amplified DNA of deceased famous people like Elvis Presley and Marilyn Monroe.^[15][16] Mullis is also a member of the USA Science and Engineering Festival's Advisory Board.^[17]

[edit] PCR and other inventions

Main articles: Taq Polymerase and History of polymerase chain reaction

In 1983, Mullis was working for Cetus Corp. as a chemist.^[13] That spring, according to Mullis, he was driving his vehicle late one night with his girlfriend, who was also a chemist at Cetus, when he had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times.^[13] Cetus took Mullis off his usual projects to concentrate on PCR full-time.^[13] Mullis succeeded on demonstrating PCR December 16, 1983.^[13] In his Nobel Prize lecture, he remarked that the success didn't make up for his girlfriend breaking up with him shortly before: "I was sagging as I walked out to my little silver Honda Civic. Neither [assistant] Fred, empty Beck's bottles, nor the sweet smell of the dawn of the age of PCR could replace Jenny. I was lonesome."^[13] He received a $10,000 bonus from Cetus for the invention.^[13]
Other Cetus scientists, including Randall Saiki and Henry Erlich, were placed on PCR projects to work on developing HIV- and other tests utilizing PCR. Saiki generated the needed data and authored the first paper to include utilization of the technique,^[4] while Mullis was still working on a paper that would describe PCR itself.^[13]
A complication at that point was that the DNA polymerase used was destroyed by the high heat used at the start of each replication cycle and had to be replaced. In 1986, Mullis started to use Thermophilus aquaticus (Taq) DNA polymerase to amplify segments of DNA. The Taq polymerase was heat resistant and would only need to be added once, thus making the technique dramatically more affordable and subject to automation. This has created revolutions in biochemistry, molecular biology, genetics, medicine and forensics.
Mullis has also invented a UV-sensitive plastic that changes color in response to light, and most recently has been working on an approach for mobilizing the immune system to neutralize invading pathogens and toxins, leading to the formation of his current venture, Altermune LLC. Mullis described this idea this way:

It is a method using specific synthetic chemical linkers to divert an immune response from its nominal target to something completely different which you would right now like to be temporarily immune to. Let's say you just got exposed to a new strain of the flu. You're already immune to alpha-1,3-galactosyl-galactose bonds. All humans are. Why not divert a fraction of those antibodies to the influenza strain you just picked up? A chemical linker synthesized with an alpha-1,3-gal-gal bond on one end and a DNA aptamer devised to bind specifically to the strain of influenza you have on the other end will link anti-alpha-Gal antibodies to the influenza virus and presto!--you have fooled your immune system into attacking the new virus.^[12][18]

[edit] Accreditation of the PCR technique

A concept similar to that of PCR had been described before Mullis' work. Nobel Prize laureate H. Gobind Khorana and Kjell Kleppe, a Norwegian scientist, authored a paper seventeen years earlier describing a process they termed "repair replication" in the Journal of Molecular Biology. Using repair replication, Kleppe duplicated and then quadrupled a small synthetic molecule with the help of two primers and DNA-polymerase. The method developed by Mullis, however, combined the use of a heat-stable polymerase and thermal cycling, which allowed the rapid and exponential amplification of large quantities of any desired DNA sequence from an extremely complex template.
The suggestion that Mullis was solely responsible for the idea of using Taq polymerase in the PCR process has been contested by his co-workers at the time, who were embittered by his abrupt departure from Cetus.^[13] However, other scientists have written that "the full potential [of PCR] was not realized" until Mullis' work in 1983,^[19] and that Mullis' colleagues failed to see the potential of the technique when he presented it to them.^[15] As a result, some controversy surrounds the balance of credit that should be given to Mullis versus the team at Cetus.^[4] In practice, credit has accrued to both the inventor and the company (although not its individual workers) in the form of a Nobel Prize and a $10,000 Cetus bonus for Mullis and $300 million for Cetus when the company sold the patent to Roche Molecular Systems. After DuPont lost out to Roche on that sale, the company unsuccessfully disputed Mullis's patent on the alleged grounds that PCR had been previously described in 1971.^[13] Mullis took Cetus' side in the case, and Khorana refused to testify for DuPont; the jury upheld Mullis's patent in 1991.^[13]
The anthropologist Paul Rabinow wrote a book on the history of the PCR method in 1996 (entitled Making PCR) in which he discussed whether or not Mullis "invented" PCR or "merely" came up with the concept of it. Rabinow, a Foucault scholar interested in issues of the production of knowledge, used the topic to argue against the idea that scientific discovery is the product of individual work, writing, "Committees and science journalists like the idea of associating a unique idea with a unique person, the lone genius. PCR is, in fact, one of the classic examples of teamwork."^[20]

[edit] Personal views

Mullis has said that the never-ending quest for more grants and staying with established dogmas has hurt science.^[13] He believes that "Science is being practiced by people who are dependent on being paid for what they are going to find out," not for what they actually produce.^[13] Mullis has been described as an "impatient and impulsive researcher" who avoids lab work and instead thinks about research topics while driving and surfing.^[21]
In his 1998 autobiography, Mullis expressed disagreement with the scientific evidence supporting climate change and ozone depletion, the evidence that HIV causes AIDS, and asserted his belief in astrology. Mullis claims climate change and the HIV/AIDS connection are due to a conspiracy of environmentalists, government agencies and scientists attempting to preserve their careers and earn money, rather than scientific evidence.^[6] Mullis has drawn controversy for his association with prominent AIDS denialist Peter Duesberg,^[7] claiming that AIDS is an arbitrary diagnosis only used when HIV antibodies are found in a patient's blood.^[8] The medical and scientific consensus is that Duesberg's hypothesis is pseudoscience, HIV having been conclusively proven to be the cause of AIDS^[22][23] and that global warming is occurring because of human activities.^[24][25][26] Seth Kalichman, AIDS researcher and author of Denying AIDS, "[admits] that it seems odd to include a Nobel Laureate among the who's who of AIDS pseudoscientists".^[9] Mullis also wrote the foreword to the book What If Everything You Thought You Knew About AIDS Was Wrong? by Christine Maggiore,^[10] an HIV-positive AIDS denialist who, along with her daughter, died of an AIDS-related illness.^[27] A New York Times article listed Mullis as one of several scientists who, after success in their area of research, go on to make unfounded, sometimes bizarre statements in other areas.^[5] An article in the Skeptical Inquirer described Mullis as an "...AIDS denialist with scientific credentials [who] has never done any scientific research on HIV or AIDS".^[11]

[edit] Use of LSD

Mullis details his experiences synthesizing and testing various psychedelic amphetamines and a difficult trip on DET in his autobiography. In a Q&A interview published in the September, 1994, issue of California Monthly, Mullis said, "Back in the 1960s and early '70s I took plenty of LSD. A lot of people were doing that in Berkeley back then. And I found it to be a mind-opening experience. It was certainly much more important than any courses I ever took."^[28] During a symposium held for centenarian Albert Hofmann, "Hofmann revealed that he was told by Nobel-prize-winning chemist Kary Mullis that LSD had helped him develop the polymerase chain reaction that helps amplify specific DNA sequences."^[29] Replying to his own postulate during an interview for BBC's Psychedelic Science documentary, "What if I had not taken LSD ever; would I have still invented PCR?" He replied, "I don't know. I doubt it. I seriously doubt it."^[30]

[edit] Extraterrestrial life

Mullis writes of having once spoken to a glowing green raccoon. Mullis arrived at his cabin in the woods of northern California around midnight one night in 1985, and, having turned on the lights and left sacks of groceries on the floor, set off for the outhouse with a flashlight. "On the way, he saw something glowing under a fir tree. Shining the flashlight on this glow, it seemed to be a raccoon with little black eyes. The raccoon spoke, saying, ‘Good evening, doctor,’ and he replied with a hello." Mullis later speculated that the raccoon ‘was some sort of holographic projection and … that multidimensional physics on a macroscopic scale may be responsible’. Mullis denies LSD having anything at all to do with this.^[31]

[edit] Personal life

Mullis enjoys surfing,^[32] and has been married three times.^[13] He has three children by two ex-wives.^[13]

[edit] Books authored

• The Polymerase Chain Reaction, 1994, with Richard A. Gibbs
• Dancing Naked in the Mind Field. 1998, Vintage Books.

Mullis's 1998 autobiography Dancing Naked in the Mind Field, gives his account of the commercial development of PCR, as well as providing insights into his opinions and experiences. In the book, Mullis chronicles his romantic relationships, use of LSD, synthesis and self-testing of novel psychoactive substances, belief in astrology and an encounter with an extraterrestrial in the form of a fluorescent raccoon.

[edit] Awards and honors

• 1990 – William Allan Memorial Award of the American Society of Human Genetics | Preis Biochemische Analytik of the German Society of Clinical Chemistry and Boehringer Mannheim
• 1991 – National Biotechnology Award | Gairdner Award | R&D Scientist of the Year
• 1992 – California Scientist of the Year Award
• 1993 – Nobel Prize in Chemistry | Japan Prize | Thomas A. Edison Award
• 1994 – Honorary degree of Doctor of Science from the University of South Carolina
• 1998 – Inducted into the National Inventors Hall of Fame ^[33] | Ronald H. Brown American Innovator Award^[34]
• 2004 – Honorary degree in Pharmaceutical Biotechnology from the University of Bologna, Italy

Mullis also received the John Scott Award in 1991, given by the City Trusts of Philadelphia to others including Thomas Edison and the Wright Brothers.^[35]

[edit] See also

• Nobel Prize controversies
• The Other Side of AIDS
• House of Numbers

[edit] References

1. ^ Shampo, M. A.; Kyle, R. A. (2002). "Kary B. Mullis--Nobel Laureate for procedure to replicate DNA". Mayo Clinic proceedings. Mayo Clinic 77 (7): 606. PMID 12108595.  edit
2. ^ Saiki, R.; Gelfand, D.; Stoffel, S.; Scharf, S.; Higuchi, R.; Horn, G.; Mullis, K.; Erlich, H. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239 (4839): 487–491. doi:10.1126/science.2448875. PMID 2448875.  edit
3. ^ ^{a b c d e} 'Biotechnology 101'' by Brian Robert Shmaefsky. Books.google.com. 2006-10-30. ISBN 9780313335280. http://books.google.com/?id=HarCo_xmABIC&pg=PA184&dq=%22kary+mullis%22. Retrieved 2010-07-27. 
4. ^ ^{a b c d e f} "Scientist at Work/Kary Mullis; After the 'Eureka', a Nobelist Drops Out" Nicholas Wade, The New York Times, September 15, 1998.
5. ^ ^{a b c} Johnson, G (2007-10-28). "Bright Scientists, Dim Notions". The New York Times. http://www.nytimes.com/2007/10/28/weekinreview/28johnson.html?_r=3&adxnnl=1&oref=slogin&ref=science&adxnnlx=1193583001-IE12EKQeJt1sjwCUOYPVWg&oref=slogin. Retrieved 2010-08-06. 
6. ^ ^{a b c d} Mullis, K (1998). Dancing Naked in the Mind Field. Vintage Books. pp. 115–118, 143–153. ISBN 0679442553. 
7. ^ ^{a b} Thomas, Charles A. (1994). "''Reason'', June 1994". Findarticles.com. http://findarticles.com/p/articles/mi_m1568/is_n2_v26/ai_15473457. Retrieved 2010-07-27. 
8. ^ ^{a b} "''Washington Informer'', May 31, 2000". Highbeam.com. 2000-05-31. http://www.highbeam.com/doc/1P1-79665692.html. Retrieved 2010-07-27. 
9. ^ ^{a b} Kalichman, Seth (2009). Denying AIDS: Conspiracy Theories, Pseudoscience, and Human Tragedy. New York: Copernicus Books (Springer Science+Business Media). pp. 177–178. ISBN 978-0-387-79475-4. 
10. ^ ^{a b} Maggiore C (2006). What If Everything You Thought You Knew About AIDS Was Wrong?. American Foundation For AIDS Alternative. ISBN 0-9674153-2-2. 
11. ^ ^{a b} Nattrass, N (2007). "AIDS Denialism vs. Science". Skeptical Inquirer 31 (5). http://www.csicop.org/si/show/aids_denialism_vs._science/. 
12. ^ ^{a b c} "Official Nobel Autobiography". Nobelprize.org. 1998-03-21. http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-autobio.html. Retrieved 2010-07-27. 
13. ^ ^{a b c d e f g h i j k l m n o p} Yoffe, Emily Emily Yoffe (Vol 122, no. 1 (July) 1994: 68–75). "Is Kary Mullis God? Nobel Prize winner's new life". Esquire. 
14. ^ "''The Economist'', 2004". Economist.com. http://www.economist.com/science/tq/displayStory.cfm?Story_id=2477036. Retrieved 2010-07-27. 
15. ^ ^{a b} ''Life on the Edge: Amazing Creatures Thriving in Extreme Environments'' by Michael Gross. Books.google.com. 2001-01-24. ISBN 9780738204451. http://books.google.com/?id=J5IwomfEGiEC&pg=PA103&dq=%22kary+mullis%22. Retrieved 2010-07-27. 
16. ^ "''The Hastings Center Report'', 1998". Questia.com. http://www.questia.com/googleScholar.qst;jsessionid=HL2GySNR8dj13v4D64WDGhlzhN8Fkspv6nJF6JwngX1NzhyfwZlK!930663388?docId=5002303854. Retrieved 2010-07-27. 
17. ^ "Advisors". Usasciencefestival.org. http://www.usasciencefestival.org/about/advisors. Retrieved 2010-07-27. 
18. ^ "Kary Mullis' next-gen cure for killer infections | Video on". Ted.com. http://www.ted.com/talks/lang/eng/kary_mullis_next_gen_cure_for_killer_infections.html. Retrieved 2010-07-27. 
19. ^ ''Artificial DNA: Methods and Applications'' by Yury E. Khudyakov, Howard A. Fields. Books.google.com. 2003. ISBN 9780849314261. http://books.google.com/?id=dexRnDtLlWUC&pg=PA20&dq=%22kary+mullis%22+%2B+%22kjell+kleppe%22. Retrieved 2010-07-27. 
20. ^ Richard Bilsker. "Ethnography of a Nobel Prize". Hyle.org. http://www.hyle.org/journal/issues/4/bilsker.htm. Retrieved 2010-07-27. 
21. ^ Fridell R (2005). Decoding life: unraveling the mysteries of the genome. Minneapolis: Lerner Publications. pp. 88. ISBN 0-8225-1196-7. 
22. ^ "Confronting AIDS: Update 1988". Institute of Medicine of the U.S. National Academy of Sciences. 1988. http://books.nap.edu/openbook.php?record_id=771&page=2. "…the evidence that HIV causes AIDS is scientifically conclusive." 
23. ^ "The Evidence that HIV Causes AIDS". National Institute of Allergy and Infectious Disease. 2009-09-04. http://www3.niaid.nih.gov/topics/HIVAIDS/Understanding/howHIVCausesAIDS/HIVcausesAIDS.htm. Retrieved 2009-10-14. ^{[dead link]}
24. ^ Oreskes, Naomi (December 2004). "BEYOND THE IVORY TOWER: The Scientific Consensus on Climate Change". Science 306 (5702): 1686. doi:10.1126/science.1103618. PMID 15576594. http://www.sciencemag.org/cgi/content/full/306/5702/1686. "Such statements suggest that there might be substantive disagreement in the scientific community about the reality of anthropogenic climate change. This is not the case. [...] Politicians, economists, journalists, and others may have the impression of confusion, disagreement, or discord among climate scientists, but that impression is incorrect." 
25. ^ "Joint Science Academies' Statement" (pdf). United States National Academies. 2005-07-06. http://nationalacademies.org/onpi/06072005.pdf. Retrieved 2011-06-09. 
26. ^ "Understanding and Responding to Climate Change" (pdf). United States National Academies. 2008. http://dels-old.nas.edu/dels/rpt_briefs/climate_change_2008_final.pdf. Retrieved 2011-06-09. 
27. ^ "Christine Maggiore, vocal skeptic of AIDS research, dies at 52". Los Angeles Times. 2008-12-30. http://www.latimes.com/news/local/la-me-christine-maggiore30-2008dec30,0,7407966.story. Retrieved 2008-12-30. 
28. ^ Schoch, Russell (September 1994). "Q&A - A Conversation with Kerry Mullis". California Monthly (Berkeley, CA: California Alumni Association) 105 (1): 20. http://www.alumni.berkeley.edu/Alumni/Cal_Monthly/September_1994/QA_-_A_Conversation_with_Kerry_Mullis.asp. Retrieved 2008-03-11. 
29. ^ Ann Harrison (2006-01-16). "LSD: The Geek's Wonder Drug?". Wired. Wired. http://www.wired.com/science/discoveries/news/2006/01/70015. Retrieved 2008-03-11. "Like Herbert, many scientists and engineers also report heightened states of creativity while using LSD. During a press conference on Friday, Hofmann revealed that he was told by Nobel-prize-winning chemist Kary Mullis that LSD had helped him develop the polymerase chain reaction that helps amplify specific DNA sequences." 
30. ^ "BBC Horizon - Psychedelic Science - DMT, LSD, Ibogaine - Part 5". BBC. 1997. http://www.youtube.com/watch?v=j2WurhYEQyY&feature=related. Retrieved 2009-10-16. 
31. ^ http://www.lrb.co.uk/v33/n22/jenny-diski/what-might-they-want Review of "The Myth and Mystery of UFOs", by Thomas Bullard
32. ^ Golden, Frederic (2000-12-13). "''Time Magazine'', December 13, 2000". Time.com. http://www.time.com/time/printout/0,8816,91819,00.html. Retrieved 2010-07-27. 
33. ^ "Hall of Fame/Inventor Profile". Invent.org. 1944-12-28. http://www.invent.org/Hall_Of_Fame/109.html. Retrieved 2010-07-27. 
34. ^ "Nobel Prize Winner Among Rondal H. Brown Award Recipients". Uspto.gov. 1998-10-13. http://www.uspto.gov/web/offices/com/speeches/98-18.htm. Retrieved 2010-07-27. 
35. ^ "John Scott Award Winners". Garfield.library.upenn.edu. 2005-10-28. http://www.garfield.library.upenn.edu/johnscottaward(full).html. Retrieved 2010-07-27. 

[edit] Further reading

• Paul Rabinow, Making PCR: a story of biotechnology (University of Chicago Press, 1996). ISBN 0-226-70147-6
• Anthony Liversidge, "Kary Mullis, the great gene machine", Omni magazine (April 1992).
• Kary Mullis, Dancing Naked in the Mind Field. Vintage Books (1998). ISBN 0679442553.

[edit] External links

Wikiquote has a collection of quotations related to: Kary Mullis

• Personal webpage
• Patent portfolio

[edit] Interviews

• 2005 interview for Nobel Prize committee
• 1998 interview with Sarah Klipfel regarding his views on HIV/AIDS

[hide] v t e Nobel Laureates in Chemistry (1976–2000) William Lipscomb (1976) Ilya Prigogine (1977) Peter D. Mitchell (1978) Herbert C. Brown / Georg Wittig (1979) Paul Berg / Walter Gilbert / Frederick Sanger (1980) Kenichi Fukui / Roald Hoffmann (1981) Aaron Klug (1982) Henry Taube (1983) Robert Merrifield (1984) Herbert A. Hauptman / Jerome Karle (1985) Dudley R. Herschbach / Yuan T. Lee / John Polanyi (1986) Donald J. Cram / Jean-Marie Lehn / Charles J. Pedersen (1987) Johann Deisenhofer / Robert Huber / Hartmut Michel (1988) Sidney Altman / Thomas Cech (1989) Elias Corey (1990) Richard R. Ernst (1991) Rudolph A. Marcus (1992) Kary Mullis / Michael Smith (1993) George Olah (1994) Paul J. Crutzen / Mario J. Molina / Frank Rowland (1995) Robert Curl / Harold Kroto / Richard Smalley (1996) Paul D. Boyer / John E. Walker / Jens Christian Skou (1997) Walter Kohn / John Pople (1998) Ahmed Zewail (1999) Alan J. Heeger / Alan MacDiarmid / Hideki Shirakawa (2000) Complete list (1901–1925) (1926–1950) (1951–1975) (1976–2000) (2001–2025)

Persondata
Name	Mullis, Kary Banks
Alternative names
Short description
Date of birth	1944-12-28
Place of birth	Lenoir, North Carolina, United States
Date of death
Place of death

 References

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2. ^ ^{a b} Saiki, R.; Scharf, S.; Faloona, F.; Mullis, K.; Horn, G.; Erlich, H.; Arnheim, N. (1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350–1354. doi:10.1126/science.2999980. PMID 2999980.  edit
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 External links

Wikimedia Commons has media related to: Polymerase chain reaction

• US Patent for PCR
• Step-through animation of PCR - Cold Spring Harbor Laboratory
• OpenWetWare
• History of the Polymerase Chain Reaction from the Smithsonian Institution Archives