Applications of PCR

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. For most, if not all of these applications, alternative methods of DNA (or RNA) amplification, can be substituted. For example helicase-dependent amplification (HDA) and recombinase polymerase amplification (RPA) are isothermal alternatives to PCR that can be used for the same purposes. This is a list of some of these applications:

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.

Infectious disease applications

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

• The human immunodeficiency virus (or HIV), 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.

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 DNA paternity 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).

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 not 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 quantitative 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.

External links

• The Reference in real time PCR -- an Academic & Industrial Information Platform
• www.eConferences.de -- Amplify your knowledge in qPCR, dPCR and NGS!

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).