© CAB International 2004
`ISSN 1466-2523
`
`Animal Health Research Reviews 5(2); 247–248
`DOI: 10.1079/AHRR200477
`
`Advances in PCR technology
`
`Lloyd H. Lauerman
`Avian Health and Food Safety Laboratory, 7612 Pioneer Way E., Washington State
`University-Puyallup, Puyallup, WA 98371–4998, USA
`
`Abstract
`
`Since the discovery of the polymerase chain reaction (PCR) 20 years ago, an avalanche of
`scientific publications have reported major developments and changes in specialized equip-
`ment, reagents, sample preparation, computer programs and techniques, generated through
`business, government and university research. The requirement for genetic sequences for
`primer selection and validation has been greatly facilitated by the development of new
`sequencing techniques, machines and computer programs. Genetic libraries, such as
`GenBank, EMBL and DDBJ continue to accumulate a wealth of genetic sequence information
`for the development and validation of molecular-based diagnostic procedures concerning
`human and veterinary disease agents. The mechanization of various aspects of the PCR
`assay, such as robotics, microfluidics and nanotechnology, has made it possible for the rapid
`advancement of new procedures. Real-time PCR, DNA microarray and DNA chips utilize
`these newer techniques in conjunction with computer and computer programs. Instruments
`for hand-held PCR assays are being developed. The PCR and reverse transcription–PCR
`(RT–PCR) assays have greatly accelerated the speed and accuracy of diagnoses of human
`and animal disease, especially of the infectious agents that are difficult to isolate or demon-
`strate. The PCR has made it possible to genetically characterize a microbial isolate
`inexpensively and rapidly for identification, typing and epidemiological comparison.
`
`Keywords: polymerase chain reaction; vaccines
`
`The polymerase chain reaction (PCR) was discovered
`by Mullis in 1983 (Mullis et al., 1986; Mullis and Faloona,
`1987) and he received a Nobel Prize for this achievement
`a decade later. Awareness of the significance of the PCR
`technique had spread throughout the scientific commu-
`nity of the world by the end of the 1980s, and scientific
`articles were being published concerning the various uses
`of the PCR technique. By 1990 there was a considerable
`pool of 16S RNA gene sequences, which had been gener-
`ated for studies of the phylogeny of microorganisms, and
`these sequences could be used for primer selection by
`sequence alignment. New
`sequencing
`techniques,
`sequencing machines and computer programs for the
`manipulation of sequence information have been devel-
`oped that have greatly accelerated the accumulation of
`
`E-mail: lhlauerman@mindspring.com
`
`genetic sequences for the development of newer and bet-
`ter PCR assays. The use of the Internet has made it
`possible to access scientific information for the compari-
`son of genetic sequences from around the world. Genetic
`libraries (GenBank, EMBL, DDBJ) continue to accumulate
`a broad selection of genetic sequence information for the
`development and validation of molecular-based diagnos-
`tic procedures concerning human and veterinary disease
`agents.
`The initial thermal cycler used in our laboratory in
`1990 was a Perkin-Elmer model 480, which had a refrig-
`eration unit to speed the rate of cooling and did not
`have heated lid technology. Nanotechnology was used
`from the beginning of these techniques. Simple com-
`puter hardware and software were built into the early
`thermal cyclers. The next generation of thermal cyclers
`had heated lids, no refrigeration (they worked off ambi-
`ent temperature, using fans), and more memory for
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`Lloyd H. Lauerman
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`programs. With the development of real-time PCR, the
`instruments became more elaborate and expensive. The
`use of standard computers and specialized computer
`software was necessary to run real-time PCR assays.
`Real-time PCR technology requires new primers and flu-
`orescent tagged probes to be developed that are much
`more expensive to prepare. The major advantage of
`real-time PCR over standard PCR assay is the one-tube
`and one-time handling, with the reading of the reaction
`during the assay.
`Initially the simple procedure of boiling was used to
`release DNA from the sample, and to obtain a greater
`yield the phenol–chloroform technique was used. Less
`toxic chemicals were researched for the release and har-
`vest of the nucleic acids. Then DNA and RNA affinity
`materials were used for the extraction of the nucleic
`acids. Initially the microcentrifuge and chemical hoods
`were the instruments necessary for DNA/RNA extraction,
`and a lot of hands-on effort was needed. Instruments
`have been developed using microfluidics, nanotechnol-
`ogy and robotics to process the samples automatically.
`Automatic sample collection is being developed using
`remote access air-samplers to evaluate environments for
`high-risk pathogens of public health importance, and for
`the routine sampling of animal environments, such as
`poultry houses, swine barns, milking parlors and food
`processing plants, for specific pathogens of economic or
`public health importance.
`One of the most important developments in PCR tech-
`nology was the discovery and use of the Taq DNA
`polymerase enzyme. By 1990, standard reagents (PCR
`buffer, dNTP, MgCl2 and Taq DNA polymerase) were
`commercially available for PCR assays. One of the limit-
`ing factors at the time was the relatively small amount of
`sequence information for specific organisms. In 1990, we
`were attempting to develop species-specific primers for
`Mycoplasma synoviae and sequenced a portion of a DNA
`probe used in Israel to detect the organism. Specific
`primers were not developed using this approach.
`However, in 1991 the last half of the 16S rRNA gene
`sequence of M. synoviae was entered into the GenBank
`by an Australian team of scientists. A complete sequence
`of M. gallisepticum 16 S rRNA gene sequence was found
`on enBank and used to perform a sequence alignment,
`which allowed us to select a set of species-specific
`primers
`for M.
`synoviae. The BLAST program
`(www.ncbi.nlm.nih.gov/BLAST/) came available, and this
`was of great assistance in rapid computer evaluation of
`primer sequences for specificity by testing them against
`all sequences in GenBank. As the years passed,
`sequence information concerning a greater number of
`
`organisms was submitted to the various genetic libraries,
`and thus we were able to develop a variety of primers
`and publish a PCR manual entitled Nucleic Acid
`Amplification Assays for Diagnosis of Animal Diseases
`(Lauerman, 1998). Recently, PCR assays have been used
`to evaluate vaccines for the presence or absence of spe-
`cific disease agents (Lauerman, 2002).
`Improvements in buffers have given greater stability
`and longer activity for reagents and enzymes, which has
`contributed to improved PCR and reverse transcrip-
`tion–PCR (RT–PCR) techniques. Since the discovery of
`Taq DNA polymerase, many improvements have been
`made, such as the synthesis of a recombinant poly-
`merase without undesirable
`side effects. Other
`polymerase enzymes have been isolated and evaluated
`from a number of thermophilic organisms having a vari-
`ety of activities different from Taq DNA polymerase.
`This allows longer segments of DNA to be produced as
`PCR amplicons with greater accuracy.
`New technology has been developed, such as DNA
`microarray and DNA chips, that gives hundreds to thou-
`sands more pieces of genetic information in a shorter
`period of time than the original PCR techniques. A
`detailed description of the DNA microarray procedure
`can be found on the web sites www.vetscite.org/
`cgi-bin/pw.exe/issue3/000035/000035.htm and http://
`www.gene-chips.com/.
`PCR technology has made it possible to genetically
`characterize microbial isolates inexpensively and rapidly
`for identification, typing and epidemiological evaluation.
`The emphasis on homeland security and bioterrorism
`preparedness in the USA has expanded the need for
`more rapid and highly accurate diagnostic capabilities to
`protect public health, animal agriculture and the associ-
`ated industries throughout the world.
`
`References
`
`Lauerman LH (1998). Nucleic acid amplification assays for diag-
`nosis of animal diseases. In: Lloyd H, editor. Annual
`Meeting, American Association of Veterinary Laboratory
`Diagnosticians, pp. 1–52.
`Lauerman LH (2002). Molecular techniques for evaluation of
`vaccines in response to national and international regula-
`tions. In: Proceedings of the United States Animal Health
`Association October 14, St Louis, MO, pp. 142–144.
`Mullis K and Faloona F (1987). Specific synthesis of DNA in
`vitro via a polymerase-catalyzed chain reaction. Methods
`in Enzymology 155: 335–350.
`Mullis K, Faloona F, Scharf S, Saiki R, Horn G and Erlich H
`(1986). Specific enzymatic amplification of DNA in vitro:
`the polymerase chain reaction. Cold Spring Harbor
`Symposia on Quantitative Biology 51: 263–273.
`
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