Understanding PCR

Understanding PCR

Remo Lobetti BVSc MMedVet (Med) PhD Dipl ECVIM

INTRODUCTION

The polymerase chain reaction (PCR) is a technique in molecular biology that analyses short sequences of DNA or RNA and is used in the diagnosis of a variety of infectious and genetic diseases. PCR is used to amplify a piece of DNA resulting a chain reaction in which the target DNA is amplified exponentially leading to numerous copies of that DNA, which can then be easily visualised on an agarose gel. By use of a reverse transcriptase step, RNA is converted to DNA and thus the technique can also be used to detect RNA (RT-PCR).

The key to understanding PCR is that every human, animal, plant, parasite, bacterium, or virus contains genetic material sequences (nucleotide sequences or pieces of DNA or RNA) that are unique to their species, and to the individual member of that species. Thus if a sample contains segments of DNA or RNA, PCR can amplify these unique sequences so they can then be used to determine with a very high probability the identity of the source (a specific person, animal, or pathogenic organism) of the trace DNA or RNA found in or on almost any sample of material.

Once the amplification is done, the amplified segments are compared to other nucleotide segments from a known source (for example, a specific person, animal, or pathogenic organism), which is done by placing PCR-generated nucleotide sequences next to known nucleotide sequences in a separating gel.

PCR IN PRACTICE

PCR can detect and identify pathogenic organisms, especially those that are difficult to identify with other methods; diagnose genetic diseases; and identify and characterise genetic mutations found in certain cancers.

PCR TECHNIQUE

PCR is done in a single tube with appropriate chemicals and a specially designed heater. The technique was developed in 1983 by Kary Mullis and Michael Smith, which resulted in them receiving a Nobel Prize 1993.

PCR utilises the following:

Sample containing a nucleotide sequence (from blood, hair, pus, skin scraping, etc.).

DNA primers, which are short single, stranded DNA that attaches to nucleotide sequences that promotes synthesis of a complementary strand of nucleotides.

DNA polymerase, which is an enzyme that, when the DNA has a primer bound, goes down the DNA segment attaching DNA building blocks to form complementary base pairs and thus synthesizes a complementary nucleotide strand of DNA.

A large excess of DNA building blocks termed nucleotides (adenine, thymidine, cytosine and guanine,) are present in solution. When these blocks are linked together, they form a nucleotide sequence or a single strand of DNA. In the solution, the single strand of DNA building blocks bind their complementary building block by weak hydrogen bonds (for example, A will only bond with T and G only with C) a complementary DNA nucleotide sequence is formed and bound to the original single stranded DNA. When the binding is completed, a complementary double strand DNA is formed in a specific sequence.

PCR begins with a segment of DNA from a sample that is placed in a tube with the reagents listed above. The solution is heated to at least 94°C; this heat breaks the hydrogen bonds that allow complementary DNA strands to form, so only single strands exist in the mixture, which is termed denaturation of double stranded DNA.

The mixture is allowed to cool to about 54°C. At this temperature, the DNA primers and DNA polymerase bind to individual single stranded DNA (termed DNA annealing). Because the building blocks are in a high concentration in the mixture, the polymerase uses them to make new complementary strands of DNA (termed DNA extension). This process creates a new double-stranded DNA molecule from each of the single strands of the original molecule.

This cycle is repeated approximately 40 times in thermal cycler, which automatically repeats the heating-cooling cycles, with the amount of each DNA sequence doubling each time the heating-cooling cycle is completed. Thus a single short segment of DNA can be amplified to about 100 billion copies after 40 doubling cycles.

TYPES OF PCR

Multiplex PCR

Multiplex PCR uses multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different segments of the DNA sequences. An important requisite for multiplex PCR is that amplicons generated must have sufficient size differences so that their base pair length can easily be differentiated when visualised by gel electrophoresis. The main use of this technique is in the simultaneous detection of numerous organisms in a single sample.

Nested PCR

Nested PCR increases the specificity of DNA amplification by reducing background due to nonspecific amplification of DNA. Two sets of primers are used in two successive PCR reactions. In the first reaction, one pair of primers is used to generate DNA products, which, in addition to the intended target, may consist of non-specifically amplified DNA fragments. These product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying longer DNA fragments than in conventional PCR, but it requires more detailed knowledge of the target sequences. For example, this technique has been used in the identification of Tritrichomonas foetus infections in cats.

Quantitative real-time PCR

This is used to simultaneously amplify and quantify a targeted DNA molecule. It enables both detection and quantification of a specific sequence in a DNA sample. Real-time PCR is a technique growing in popularity in veterinary medicine. Its uses include the blood detection of Babesia species, Leishmania, and the simultaneous detection of multiple faecal helminth infections.

CANINE GENETIC DISEASE TESTING

Over the past few years, major advances have been made in the understanding of the molecular basis for inherited diseases in dogs. Most of the tests, available today have been developed using a comparative medicine approach to canine genetics, which utilises previously identified human diseases similar to the canine ones and tests the gene or genes responsible for the human disease to see if they are also mutated in the canine disease. Another approach is to identify genes that cause diseases in dogs, which have not previously been implicated in the equivalent human diseases. The latter approach is based that human populations tend to be more outbred than purebred dogs, which makes genetic analysis easier in dogs then in people.

There are two different types of tests available for DNA genetic testing, namely the direct and the indirect DNA test. Each of these different types of tests has their own sources of error and therefore has different implications for breeding decisions. Both of these types of testing could be used for clinical diagnosis of diseases when normal diagnostic tools might be invasive and carry with them life-threatening complications. Presently DNA based genetic tests are available for over 50 inherited diseases in dogs and the number of tests increases all the time.

Direct test

This test is designed to assay the change in DNA sequence in a particular gene that leads to a disease and can be done using an EDTA blood sample or from a cheek swab.

Indirect test

This test uses DNA markers referred to as microsatellite markers, which are small pieces of DNA sequence that contain repeats of 2-4 nucleotides and are excellent tools for individual identification, parentage analysis and for linkage analysis to a disease gene. The test is indirect since the disease-causing gene is unknown and the status of the disease-causing gene; mutant or normal, is inferred from the microsatellite markers. There is also a higher error rate associated with them compared to a direct DNA test. Once a linked microsatellite for a particular disease is identified in one species, the equivalent regions in the genomes of other species are located and candidate genes investigated. If there are no candidate genes based on comparison to other species, then the area near the disease gene is narrowed by checking additional nearby microsatellites. The techniques used to identify a gene without candidates can take many years and can be very expensive.

INFECTIOUS DISEASE DIAGNOSTICS

PCR has changed the clinical practice of infectious disease medicine and with advancement of technology may replace traditional culture-based assays for many animal pathogens. PCR has a wide range of clinical applications including pathogen detection, evaluation of emerging novel infections, and surveillance of infectious disease prevalence in a population.

PCR  assays  are of great value for documentation of infections that are difficult to culture, (Ehrlichia and Mycoplasma spp.) or cannot be cultured (Haemoplasmosis). Specificity can be very high, depending on the primers used in the reaction. Primers can be designed to detect one genus but not others or designed to identify only one species. For example, PCR can detect all Haemoplasmas within a blood sample or just one species such as Mycoplasma haemofelis.

Although PCR can be a highly sensitive test, a positive result does not always prove that the infection is resulting in clinical illness:

As PCR detects DNA of both live and dead organisms, a positive result may be achieved even if the infection has been controlled.

When the organism commonly infects the background population of healthy animals, interpretation of results for a single animal can be difficult. For example, Bartonella henselae can infect up to 20% of healthy cats and so a positive result in a clinically ill cat does not prove a disease association.

PCR cannot discriminate between vaccine strains and field strains, thus a positive result does not indicate presence of a pathogenic strain.

Real-time PCR can be used to determine the amount of microbial DNA in a sample and thus possible that the DNA load will correlate to the presence of disease. However, some agents are very host adapted and can have large amounts of DNA present in samples from healthy carrier cats. For example, the number of M. haemominutum copy numbers per µl of blood does not correlate to the haematocrit.

False positives in PCR testing can occur for a variety of reasons. In clinical settings, background contamination is a common cause of false positive reactions. The predominant source of contamination is derived from “carry over” products from previous PCR reactions. Unless a great deal of care is exercised, these products can contaminate reagents, tubes, pipettes, laboratory surfaces and even clothing.

False negative results can occur if the concentration of the pathogen is very low in the sample. This can be overcome by using methods to concentrate and/or purify the sample before initiating the PCR reaction.

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