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PCR

Polymerase chain reaction (PCR)

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Figure 1. Schematic depiction of a single qPCR cycle. Shown above are: a) The denaturation stage when double-stranded DNA is separated into single strands (ssDNA) at high temperature. b) The annealing stage where the temperature is lowered, a primer binds to the three prime (3’) end of the specific sequence targeted for amplification, and (in the case of qPCR) a probe with a fluorescent reporter and quencher binds to the 5’ end. c) The elongation stage where the polymerase enzyme duplicates the sequence by attaching to the primer to start building a new complementary strand until it finishes by dissociating the probe and enabling the fluorescent signal.

DNA evidence in forensics has cracked many crimes. However, there were circumstances where the amount of the collected DNA at the crime scene was too small for the conventional techniques to generate a reliable profile. Polymerase chain reaction, invented by Kary Mullis in 1983, has revolutionized not only the science of forensics but also the entire field of molecular biology and medicine by allowing us to chemically create billions of copies of a single DNA strand.

First, denaturation (Fig. 1) begins by heating a strand of DNA - mixed together with deoxyribonucleoside triphosphates (dNTPs), primers, Taq polymerase, and buffer - to a temperature of about 98℃ so that the two chains forming the double helix separate. The result is the formation of two DNA templates. In the next stage, called annealing, the temperature is lowered to 48℃-72℃, allowing DNA templates to combine with DNA primers (single-stranded nucleic acid).

DNA primers are short, single-stranded DNA sequences that attach at one end of each template and are very specific to the DNA intended for amplification. In the next stage, called extension or elongation, the temperature is about 68℃-72℃, and the primer recruits Taq polymerase to the site to add dNTPs to the templates forming replicas. These three steps are repeated, and the amplification is exponential so that the number of copies equals 2(N+1), where N is the number of cycles. If N=25, the number of copies is about 67 million.

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Figure 2. Amplification plot. Typical amplification charts of nucleic acid targets are generated by plotting the relative fluorescence signal detected by the sensor(s) as a function of the number of cycles. These charts usually include a series of dilutions in initial concentration of the amount of target added to the reaction. Once above the fluorescence threshold intensity, the signal increases exponentially then linearly as reaction reagents are consumed, finally reaching a plateau level.

A certain subset of PCR, real-time or quantitative PCR (qPCR) employs a fluorescent photonic technique called Forster resonance energy transfer (FRET) by adding an additional molecule into the mix, a probe (Fig.1). Probes are short complementary sequences, also very specific to the intended target DNA, but with the addition of a fluorescent reporter dye and a FRET quencher in close proximity. Instead of binding at the beginning of the target DNA strand, the probe binds at the end, and, as the Taq polymerase finishes its replication, it cleaves the probe and destroys the FRET quenching relationship, allowing the fluorescent dye to report the duplicate strand to fluorescence detectors like cameras and silicon photodiodes. The resulting fluorescence intensity increases with the number of cycles in relation to the initial concentration of target in the sample (Fig. 2).

In this example of photonics applied to molecular biology, the quantity of DNA amplified can be monitored and detection of specific DNA or RNA needed for molecular diagnostic tests (Mol Dx) is possible. For example, testing for active infections in COVID-19 cases employs reverse transciptase quantitative PCR (RT-qPCR). The virus is a positive-sense, single-stranded RNA (ribonucleic acid) type, using the RNA as the genetic material. Once inside a host, the virus uses its RNA to hijack the cell’s own protein machinery to disrupt normal cellular functions and replicate itself.

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Figure 3. Schematic of the transcription and translation process. Encoded in DNA is the recipe for all biological function. The process of DNA replication occurs naturally, for example when cells divide, and can also be used as a tool to amplify the DNA. Transcription is the process in which the genetic code in DNA is read and transcribed into the complementary RNA molecule with the help of an enzyme, RNA polymerase. Different enzymes, called reverse transcriptases, can also invert this process and create DNA from RNA. Finally, translation is the process of creating proteins, the workhorse molecules in cells and organisms.

The reverse transcriptase term simply means the RNA is reverse transcribed (Fig. 3) back into complementary DNA (cDNA) prior to amplification by qPCR. Small quantities of viral genetic material in swab and saliva tests are undetectable even by the most sensitive photonic detectors, but when the genetic material is amplified millions of times, we can see the presence of the viral RNA. A key factor in the limit of detection (LOD) (Fig. 2) is more sensitive and wider dynamic range detectors, along with improvements in chemical reagents.

Lastly, digital PCR (dPCR) is yet another subset of this DNA amplification technology that employs photonic detectors to measure fluorescence similar to qPCR. However, in digital PCR, samples are split into thousands of reactions to detect and quantify exact copy numbers of the original targets. For example, dPCR is useful when true viral load counts or absolute quantities of specific genetic material need to be obtained. Due to the small volumes inherent in splitting a sample into thousands of separate reactions versus measuring the larger aggregate reaction, dPCR very often requires highly sensitive photodetectors, like silicon photomultipliers (SiPMs/MPPCs) and photomultiplier tubes (PMTs), capable of detecting even single photons from the fluorescent reporters.

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