Discovery and development of PCR
Polymerase chain reaction (PCR) is an in vitro diagnostic technique that can replicate or amplify target DNA billions of times in an hour or less. The amplification reaction is primer-mediated. Oligonucleotide primers anneal to the DNA region to be amplified and located on both sides of it. DNA gathering enzyme adds the nucleotide substrate to the 3′ end of the primer to extend and synthesize new DNA chain. After many thermal cycles, a sufficient amount of DNA is synthesized for subsequent testing, analysis, and manipulation. PCR is usually used in clinical diagnosis and basic scientific experimental research, because of its excellent sensitivity and specificity. PCR has quickly become a standard method of diagnostic microbiology and a routine method of gene sequence analysis. PCR was developed by Kary Mullis in 1983 and he won the Nobel Prize in Chemistry in 1993. Since the development of PCR research and diagnostic applications in the 1990s, clinical microbiologists still rely heavily on laboratory-developed PCR testing methods to detect many important sources of infection, such as HIV detection, HPV detection and COVID-19 detection.
Principle of PCR
Polymerase chain reaction (PCR) is a detection technology established based on DNA double helix and DNA replication in vivo. The double helix of DNA is stabilized by hydrogen bonds established between the nitrogenous bases present on the two strands. The four nucleotide bases that make up DNA are adenine (A), cytosine (C), guanine (G), and thymine (T). All four bases have a heterocyclic structure, but structurally, adenine and guanine are derivatives of purine and are called purine bases, while cytosine and thymine are related to pyrimidines and are called pyrimidine bases. DNA replication refers to the process of duplicating DNA double strands before cell division. The result of duplication is that one double strand becomes two identical double strands, and each double strand is the same as the original double strand. This process is successfully completed through a mechanism called semi-reserved replication. (see Figure 1)
In the initial stage of PCR, the high temperature unwinds the DNA double-strands, and the oligonucleotide primers bind to the target single-stranded DNA at the annealing temperature. After the generation of DNA fragments, on the basis that the primer provides the 3′-OH end, the DNA polymerase catalyzes the replication process of the two strands of DNA at the same time, until the synthesis reaches the next high temperature melting and enters the next cycle. In each cycle, the target DNA is theoretically doubled, and the target molecule is amplified by 2n times in n cycles. Based on this principle, a variety of PCR-related detection technologies have been established, such as real-time PCR, allele-specific PCR, degenerate PCR, nested and Heminested PCR, multiplex PCR and reverse transcription PCR. (see Figure 2)
Nucleotides as PCR substrates
An important discovery that makes PCR a routine basic research and clinical diagnostic tool is thermostable DNA polymerase. The heat-stable DNA polymerase is isolated from Thermus aquaticus that inhabits hot springs at temperatures exceeding 90°C. This enzyme is called Taq DNA polymerase, and it remains active despite repeated heating during multiple amplification cycles. In addition to DNA polymerase, the basic components of PCR include oligonucleotide primers, deoxynucleoside triphosphates (dNTPs), divalent cations (such as magnesium chloride), target DNA, and buffers. Primers are oligonucleotides, usually 20-25 bases long, to identify the specific sequence of the target. At a specific annealing temperature, two primers bind to the end of the region, and each primer binds to the complementary strand of the target DNA. The length and sequence of the primer determine its annealing temperature. After annealing, the primer will form a binding site for DNA polymerase. This short double-stranded part initiates the DNA replication or amplification process. dNTP is a substrate for the synthesis of DNA strands or amplicons. The dNTP mixture includes dATP, dCTP, dGTP, and dTTP, usually in equimolar concentrations. Uracil N-glycosylase (UNG) is usually added in clinical testing PCR to prevent aerosol pollution, because in the synthesis product where dUTP is added instead of dTTP, dUTP is synthesized into DNA, and UNG can specifically degrade dUTP-DNA molecule. In addition, in order to increase the specificity and sensitivity of PCR, some modified nucleotide substrates are used in PCR detection, for example, dNTPαS and dNTPαSe.
References
1. Mullis K B, Faloona F A. [21] Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction[J]. Methods in enzymology, 1987, 155: 335-350.
2. Saiki R K, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia[J]. Science, 1985, 230(4732): 1350-1354.
3. Saiki R K, Gelfand D H, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase[J]. Science, 1988, 239(4839): 487-491.
4. Fredricks D N, Relman D A. Application of polymerase chain reaction to the diagnosis of infectious diseases[J]. Clinical infectious diseases, 1999: 475-486.
5. Marlowe E M, Novak-Weekley S M, Cumpio J, et al. Evaluation of the Cepheid Xpert MTB/RIF assay for direct detection of Mycobacterium tuberculosis complex in respiratory specimens[J]. Journal of clinical microbiology, 2011, 49(4): 1621-1623.
6. Selvaraju S B, Wurst M, Horvat R T, et al. Evaluation of three analyte-specific reagents for detection and typing of herpes simplex virus in cerebrospinal fluid[J]. Diagnostic microbiology and infectious disease, 2009, 63(3): 286-291.
7. Sire J M, Vray M, Merzouk M, et al. Comparative RNA quantification of HIV-1 group M and non-M with the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 v2. 0 and Abbott Real-Time HIV-1 PCR assays[J]. JAIDS Journal of Acquired Immune Deficiency Syndromes, 2011, 56(3): 239-243.
8. Tang Y W, Procop G W, Persing D H. Molecular diagnostics of infectious diseases[J]. Clinical chemistry, 1997, 43(11): 2021-2038.
9. Kopp M U, De Mello A J, Manz A. Chemical amplification: continuous-flow PCR on a chip[J]. Science, 1998, 280(5366): 1046-1048.
10. Pang J, Modlin J, Yolken R. Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA[J]. Molecular and cellular probes, 1992, 6(3): 251-256.
11. Rand K H, Rampersaud H, Houck H J. Comparison of two multiplex methods for detection of respiratory viruses: FilmArray RP and xTAG RVP[J]. Journal of Clinical Microbiology, 2011, 49(7): 2449-2453.