Biochemistry, Polymerase Chain Reaction

Fundamentals

The main components of PCR are a template, primers, free nucleotide bases, and the DNA polymerase enzyme. The DNA template contains the specific region of interest for amplification, such as DNA extracted from a piece of hair. Primers, or oligonucleotides, are short single strands complementary to each DNA strand of the target region. Both forward and reverse primers are required, one for each complementary strand of DNA. DNA polymerase is the enzyme that carries out DNA replication. Thermostable analogs of DNA polymerase I, such as Taq polymerase, initially found in a bacterium that grows in hot springs, are a common choice due to their resistance to the heating and cooling cycles necessary for PCR.[1]

PCR takes advantage of the DNA complementary base pairing, double-stranded structure, and melting temperature of DNA molecules. This process involves cycling through 3 sequential rounds of temperature-dependent reactions: DNA melting (denaturation), annealing, and enzyme-driven DNA replication (elongation). Denaturation begins by heating the reaction to about 95°C, disrupting the hydrogen bonds that hold the two strands of template DNA together. Next, the reaction's temperature is reduced to around 50 to 65°C, depending on the physicochemical variables of the primers, enabling the annealing of complementary base pairs.[2] 

The primers, added to the solution in excess, bind to the beginning of the 3' end of each template strand and prevent re-hybridization of the template strand with itself. Lastly, enzyme-driven DNA replication begins by setting the reaction temperature to the point that optimizes DNA polymerase enzyme activity, around 75 to 80°C. At this point, DNA polymerase, which needs double-stranded DNA to begin replication, synthesizes a new DNA strand by assembling free nucleotides in solution in the 5' to 3' direction to produce two complete sets of complementary strands. The newly synthesized DNA is now identical to the template and will be used as such in the progression of PCR cycles.

Given that previously synthesized DNA strands serve as templates, DNA amplification using PCR increases exponentially, where the copies of DNA double at the end of each replication step. The exponential replication of the target DNA eventually plateaus around 30 to 40 cycles, mainly due to reagent limitation. This plateau can also be due to inhibitors of the DNA polymerase enzyme activity found in the sample, self-annealing of the accumulating product, and accumulation of pyrophosphate molecules.[3]

Real-Time PCR

Initially, PCR technology was limited to qualitative and semi-quantitative analysis due to the limitations of nucleic acid quantification. At that time, the DNA product was separated by size via agarose gel electrophoresis to verify if the target gene was amplified successfully. Ethidium bromide, a molecule that fluoresces when bound to dsDNA, was used to estimate the amount of DNA by comparing the intensity of separated bands but was not sensitive enough for rigorous quantitative analysis.

Improvements in fluorophore development and instrumentation led to thermocyclers that no longer required measurement of only end-product DNA. This process, known as real-time or quantitative PCR (qPCR), has allowed for detecting dsDNA during amplification. The qPCR thermocyclers can excite fluorophores at specific wavelengths, detect their emission with a photodetector, and record the values. The sensitive collection of numerical values during amplification has strongly enhanced quantitative analytical power.

The sensitivity of fluorophores has been an essential aspect of qPCR development. Two main types of fluorophores are used in qPCR: those that bind specifically to a given target sequence and those that lack specificity in binding. One of the most effective and widely used non-specific markers, SYBR Green, after binding to the minor groove of dsDNA, exhibits a 1000-fold increase in fluorescence compared to being free in solution. However, if even more specificity is desired, a sequence-specific oligonucleotide, or hybridization probe, can be added, which binds to the target gene at some point, following the primer (after the 3' end). These hybridization probes contain a reporter molecule at the 5' end and a quencher molecule at the 3' end. The quencher molecule effectively inhibits the reporter from fluorescing while the probe is intact. However, upon contact with DNA polymerase I, the hybridization probe is cleaved, allowing the dye's fluorescence.

Reverse-Transcription PCR 

The PCR technology has been creatively expanded, and reverse-transcription PCR (RT-PCR) is one of the most significant advances. Real-time PCR is frequently confused with reverse-transcription PCR, but these are different techniques. In RT-PCR, the DNA amplified is derived from mRNA using reverse-transcriptase enzymes, DNA polymerases expressed by RNA-containing retroviruses, to generate a complementary DNA (cDNA) library. Using primer sequences for genes of interest, traditional PCR methods can be used with the cDNA to study the expression of genes qualitatively. Currently, reverse-transcription PCR is commonly used with real-time PCR, which allows one to quantitatively measure the relative change in gene expression across different samples.

Issues of Concern

One of the main disadvantages of PCR technology is that PCR reaction is susceptible to contamination. Trace amounts of RNA or DNA contamination in the sample can produce highly misleading results. Another disadvantage is that the primers designed for PCR require sequence data and, therefore, can only be used to identify the presence or absence of a known pathogen or gene. Also, sometimes the primers used for PCR can anneal non-specifically to sequences that are similar, but not identical, to the target gene.[4]

Another potential issue of using PCR is the possibility of primer dimer (PD) formation. PD is a potential by-product and consists of primer molecules that have hybridized with each other due to the strings of complementary bases in the primers. The DNA polymerase amplifies the PD, leading to competition for PCR reagents that could be used to amplify the target sequences.[5]

Clinical Significance

PCR amplification is an indispensable tool with various applications within medicine. Often, the process is used to test for the presence of specific alleles, such as prospective parents screening for genetic carriers. PCR amplification can also be used to diagnose the presence of disease directly and for mutations in the developing embryo. For example, the first time PCR was used in this way was for the diagnosis of sickle cell anemia through the detection of a single gene mutation.[6]

Additionally, PCR has dramatically revolutionized the diagnostic potential for infectious diseases; it can rapidly determine the identity of microbes that were traditionally unable to be cultured or that required weeks for growth.[7] Pathogens routinely detected using PCR include Mycobacterium tuberculosis, human immunodeficiency virus, herpes simplex virus, syphilis, etc. Moreover, qPCR is used to test the qualitative presence of microbes and quantify the bacterial, fungal, and viral loads.[8]

The sensitivity of diagnostic tools for mutations of oncogenes and tumor suppressor genes has been improved at least 10,000-fold due to PCR, allowing for earlier diagnosis of cancers like leukemia. PCR has also enabled more nuanced and individualized therapies for cancer patients. Additionally, PCR can be used for the tissue typing vital to organ implantation and has even been proposed as a replacement for antibody-based tests for blood type. PCR also has clinical applications in prenatal testing for genetic diseases and clinical pathologies. Samples are obtained either via amniocentesis or chorionic villus sampling.[9] 

In forensic medicine, short pieces of repeating, highly polymorphic DNA, coined short tandem repeats (STRs), are amplified and used to compare specific gene variations to differentiate individuals.[10] Primers that are specific to the loci of these STRs are used and amplified using PCR. Various loci contain STRs in the human genome, and the statistical power of this technique is enhanced by checking multiple sites.