PCR Technology: Key Milestones in Development and Maturation

Illustration showing a double helix of DNA on a dark background with text outlining significant achievements in PCR by decade.

Polymerase Chain Reaction (PCR) technology has advanced molecular biology and diagnostics, enabling scientists to amplify and analyze fragments of DNA with precision and efficiency. PCR evolved to become a fundamental tool in genetic research, medical diagnostics, forensic science, and biotechnology.

Scientific publications have been pivotal in the adoption and advancement of PCR as a cornerstone of molecular biology. See the key publications in this article. For a quick read, check out the infographic: PCR at 40.

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Invention of PCR

The concept of DNA amplification was a groundbreaking idea in the early 1980s. The challenge was to develop a method that could replicate specific DNA sequences. In 1983, Kary Mullis, a biochemist at Cetus Corporation, invented PCR. His vision was to use a cyclical process of heating and cooling to denature DNA, anneal primers, and extend new DNA strands, thus amplifying the target sequence. This proof of concept formed the basis of PCR, for which Kary Mullis was awarded the Nobel Prize in Chemistry, 1993.

In 1985, Mullis and his colleagues published the first paper describing PCR. This publication, along with the filing of a patent, marked the formal introduction of PCR to the scientific community and sparked interest in its potential applications.

Technological innovations that advanced PCR

The drawback of Mullis’ experiment was the denaturation of DNA polymerases from high temperature cycles during thermal cycling. A watershed moment for PCR was the introduction of Taq polymerase, an enzyme derived from the thermophilic bacterium Thermus aquaticus. Taq polymerase is heat-stable, allowing it to withstand the high temperatures.

Automated thermal cyclers that allowed precise control of temperature cycles were developed in the 1990s. The invention of thermal cyclers coupled with heat stable Taq polymerases greatly improved the efficiency and reliability of PCR, driving further adoption.

Find out more about the history of thermal cyclers
Learn more about the advancements in temperature optimization in thermal cyclers

Innovations and variations of PCR

Real-Time PCR (qPCR) combines PCR amplification of DNA fragments and real-time monitoring of PCR products. By using fluorescent dyes or probes, scientists could quantify the amount of DNA produced during each cycle. RT-qPCR is widely used in diagnostics and research, where precise quantification is necessary.

Learn more on the essentials of qPCR

Reverse Transcription PCR (RT-PCR) combines the principles of reverse transcription and PCR for RNA analysis. The reverse transcription step converts RNA into complementary DNA (cDNA), which is amplified by PCR. RT-PCR has been vital for gene expression studies and RNA virus detection, such as HIV and SARS-CoV-2.

Discover one-step and two-step RT-PCR

Digital PCR is a more recent innovation that allows for the absolute quantification of DNA molecules by partitioning the sample into numerous individual reactions. This method offers higher precision and sensitivity, making it valuable for applications requiring accurate DNA quantification.

Learn more about digital PCR
Find out more about the Common PCR methods and their core benefits

PCR in modern research and diagnostics

PCR has become indispensable in genetic research, enabling the amplification and sequencing of specific DNA regions. In medical diagnostics, PCR is used to detect infectious agents, identify genetic mutations, and monitor disease progression. Forensic science has also benefited from PCR, allowing analysis of minute DNA samples from crime scenes.

The COVID-19 pandemic put PCR technology at the forefront of global healthcare. PCR tests for SARS-CoV-2 became widely used for diagnosing COVID-19, enabling rapid and accurate detection of the virus. The pandemic spurred innovations in nucleic acid amplification technologies, including faster and more scalable isothermal amplification methods, which were essential for managing the public health crisis.

Learn more about isothermal amplification
Discover the wide-ranging applications of PCR

Since its invention, PCR has been an engine transforming science and medicine. Ongoing advancements such as innovations in high fidelity enzymes and sophisticated instrumentation are improving the speed, accuracy, and versatility of PCR. As we look to the future, PCR will continue to be a chief cornerstone in advancing molecular biology.

Landmark publications that propelled PCR technology are listed below.

Year	Milestone	Reference
1983	PCR invented Kary Mullis, working at Cetus, creates PCR to synthesize DNA from a single, specific location in the genome.	The Nobel Prize. 2025.
1985	First publication of PCR First description of the PCR process.	Saiki RK, Scharf S, Faloona F, et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 230(4732):1350–1354.
1987	First thermal cycler introduced Cetus Corporation introduced the first thermal cycler (T1C), a crucial innovation for standardizing PCR.	30 Years of Thermal Cycler Innovations
1988	Taq polymerase introduced Introduced the use of Taq polymerase.	Randall RK, Gelfand DH, Stoffel S, et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491.
1988	RT-PCR demonstrated Presented the concept of combining reverse transcription and PCR, particularly for amplifying rare RNA transcripts, making it a foundational reference for RT-PCR technology.	Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.Proc Natl Acad Sci U S A 85(23):8998–9002.
1988	Multiplex PCR demonstrated Simultaneous amplification of multiple targets in a single reaction by using multiple primer pairs, significantly improving throughput.	Chamberlain JS, Gibbs RA, et al. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.Nucleic Acids Res 16(23):11141–11156.

Year	Milestone	Reference
1991	First high-fidelity polymerase High-fidelity polymerase described to reduce error rates and help drive accurate PCR sequencing and cloning.	Mattila P, Korpela J, Tenkanen T, et al. (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase-an extremely heat stable enzyme with proofreading activity.Nucleic Acids Res 19(18):4967–4973.
1995	DNA sequencing Publication of the first completely sequenced genome of Haemophilus influenza using the GeneAmp PCR System 9600. Whole-genome random sequencing dramatically accelerated large genome sequencing projects, including the Human Genome Project.	Fleischmann RD, Adams MD, White O, et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512.
1996	qPCR invented Development of fluorescence-based detection systems, such as SYBR Green and TaqMan probes, enabled real-time monitoring of DNA amplification and paved the way for quantitative molecular biology.	Heid CA, Stevens J, Livak KJ, et al. (1996) Real time quantitative PCR. Genome Res 6(10):986–994.
1996	RT-qPCR demonstrated Introduced the real-time monitoring of DNA amplification using fluorescence-based detection. Although focused on qPCR, it established the platform and methodology that would also underpin RT-qPCR for quantifying RNA.	Gibson UE, Heid CA, Williams PM, et al. (1996) A novel method for real time quantitative RT-PCR. Genome Res 6(10):995–1001.
1996	First qPCR instrument ABI PRISM 7700: Integrated fluorescence detection system for real-time monitoring of DNA amplification. This instrument enabled researchers to quantify DNA and RNA in real-time, paving the way for widespread applications in diagnostics, gene expression analysis, and more.	Desjardin LE, Chen Y, Perkins MD, et al. (1998) Comparison of the ABI 7700 System (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis. J Clin Microbiol 36(7):1964–1968.

Year	Milestone	Reference
2000	Isothermal amplification invented Described the principles of LAMP and demonstrated its effectiveness as an isothermal amplification method.	Notomi T, Okayama H, Masubuchi H, et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):63.
2001	dPCR invented Introduced the term "digital PCR" and demonstrated its principle of partitioning and absolute quantification of nucleic acids. It is considered the foundational publication for dPCR.	Vogelstein B, Kinzler KW (1999) Digital PCR.Proc Natl Acad Sci U S A 96(16):9236–9241.
2002	Maturation of multiplex qPCR Fluorescent probe chemistries, such as TaqMan and Molecular Beacons, and qPCR instrumentation were further optimized to enable multiplexing, allowing reliable simultaneous quantification of multiple targets in a single reaction.	Whitcombe D, Theaker J, Guy SP, et al. (1999) Detection of PCR products using self-probing amplicons and fluorescence.Nat Biotechnol 17(8):804–807.
2005	NGS sequencing demonstrated Introduced the first commercially available NGS technology, the 454 pyrosequencing platform, which utilized massively parallel sequencing of DNA fragments in picolitre-sized wells.	Margulies M, Egholm M, Altman WE, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors.Nature 437(7057):376–380.
2009	MIQE guidelines first published Established guidelines for conducting and reporting PCR to help enable PCR reliability and reproducibility between labs.	Bustin SA, Benes V, Garson JA, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.Clin Chem 55(4):611–622.

Year	Milestone	Reference
2011	dPCR commercialized First dPCR instrument available in market.	Butkus B (2010) Digital PCR Space Heating up as Life Science Tool Vendors Begin Staking Claims.genomeweb
2012	CRISPR-Cas9 discovered Demonstrated the ability to program the Cas9 protein with guide RNAs to target specific DNA sequences for cleavage, marking the development of CRISPR-Cas9 as a genome-editing tool.	Jinek M, Chylinski K, Fonfara I, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337(6096):816–821.
2016	Digital PCR demonstrated for low-abundance targets Highlights the advantages of digital PCR by pairing the QuantStudio 3D Digital PCR System and ProFlex Thermal Cycler; digital PCR improves mitochondrial DNA deletion detection.	Belmonte FR, Martin JL, Frescura K et al. (2016) Digital PCR methods to improve detection sensitivity and measurement precisions of low-abundance mtDNA deletions. Sci Rep 6:25186.

Year	Milestone	Reference
2020	PCR becomes a household term COVID pandemic brings PCR into the public vernacular and household discussions.	Velavan TP, Meyer CG (2020) COVID-19: A PCR-defined pandemic.Int J Infect Dis 103:278–279.
2022	cfDNA as a non-invasive biomarker Digital PCR provides sensitivity and specificity to use cell-free DNA as a non-invasive biomarker.	Edwards RL, Menteer J, Lestz RM, et al. (2022) Cell-free DNA as a solid-organ transplant biomarker: technologies and approaches.Biomark Med 16(5):401–415.
2024	Implications of sex in disease explored Loss of Y chromosome on cell studied to help understand the implications of sex related to disease.	Celli L, Gasparini P, Biino G et al. (2024) CRISPR/Cas9 mediated Y-chromosome elimination affects human cells transcriptome.Cell Biosci 14(1):15.
2024	Advances in prime editing Prime editing, using a reverse transcriptase enzyme, advances to improve cellular gene editing capabilities and accuracy.	Yan J, Oyler-Castrillo P, Ravisankar P et al. (2024) Improving prime editing with an endogenous small RNA-binding protein. Nature 628:639–647.

1980s

Year	Milestone	Reference
1983	PCR invented Kary Mullis, working at Cetus, creates PCR to synthesize DNA from a single, specific location in the genome.	The Nobel Prize. 2025.
1985	First publication of PCR First description of the PCR process.	Saiki RK, Scharf S, Faloona F, et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 230(4732):1350–1354.
1987	First thermal cycler introduced Cetus Corporation introduced the first thermal cycler (T1C), a crucial innovation for standardizing PCR.	30 Years of Thermal Cycler Innovations
1988	Taq polymerase introduced Introduced the use of Taq polymerase.	Randall RK, Gelfand DH, Stoffel S, et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491.
1988	RT-PCR demonstrated Presented the concept of combining reverse transcription and PCR, particularly for amplifying rare RNA transcripts, making it a foundational reference for RT-PCR technology.	Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.Proc Natl Acad Sci U S A 85(23):8998–9002.
1988	Multiplex PCR demonstrated Simultaneous amplification of multiple targets in a single reaction by using multiple primer pairs, significantly improving throughput.	Chamberlain JS, Gibbs RA, et al. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.Nucleic Acids Res 16(23):11141–11156.

1990s

Year	Milestone	Reference
1991	First high-fidelity polymerase High-fidelity polymerase described to reduce error rates and help drive accurate PCR sequencing and cloning.	Mattila P, Korpela J, Tenkanen T, et al. (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase-an extremely heat stable enzyme with proofreading activity.Nucleic Acids Res 19(18):4967–4973.
1995	DNA sequencing Publication of the first completely sequenced genome of Haemophilus influenza using the GeneAmp PCR System 9600. Whole-genome random sequencing dramatically accelerated large genome sequencing projects, including the Human Genome Project.	Fleischmann RD, Adams MD, White O, et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512.
1996	qPCR invented Development of fluorescence-based detection systems, such as SYBR Green and TaqMan probes, enabled real-time monitoring of DNA amplification and paved the way for quantitative molecular biology.	Heid CA, Stevens J, Livak KJ, et al. (1996) Real time quantitative PCR. Genome Res 6(10):986–994.
1996	RT-qPCR demonstrated Introduced the real-time monitoring of DNA amplification using fluorescence-based detection. Although focused on qPCR, it established the platform and methodology that would also underpin RT-qPCR for quantifying RNA.	Gibson UE, Heid CA, Williams PM, et al. (1996) A novel method for real time quantitative RT-PCR. Genome Res 6(10):995–1001.
1996	First qPCR instrument ABI PRISM 7700: Integrated fluorescence detection system for real-time monitoring of DNA amplification. This instrument enabled researchers to quantify DNA and RNA in real-time, paving the way for widespread applications in diagnostics, gene expression analysis, and more.	Desjardin LE, Chen Y, Perkins MD, et al. (1998) Comparison of the ABI 7700 System (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis. J Clin Microbiol 36(7):1964–1968.

2000s

Year	Milestone	Reference
2000	Isothermal amplification invented Described the principles of LAMP and demonstrated its effectiveness as an isothermal amplification method.	Notomi T, Okayama H, Masubuchi H, et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):63.
2001	dPCR invented Introduced the term "digital PCR" and demonstrated its principle of partitioning and absolute quantification of nucleic acids. It is considered the foundational publication for dPCR.	Vogelstein B, Kinzler KW (1999) Digital PCR.Proc Natl Acad Sci U S A 96(16):9236–9241.
2002	Maturation of multiplex qPCR Fluorescent probe chemistries, such as TaqMan and Molecular Beacons, and qPCR instrumentation were further optimized to enable multiplexing, allowing reliable simultaneous quantification of multiple targets in a single reaction.	Whitcombe D, Theaker J, Guy SP, et al. (1999) Detection of PCR products using self-probing amplicons and fluorescence.Nat Biotechnol 17(8):804–807.
2005	NGS sequencing demonstrated Introduced the first commercially available NGS technology, the 454 pyrosequencing platform, which utilized massively parallel sequencing of DNA fragments in picolitre-sized wells.	Margulies M, Egholm M, Altman WE, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors.Nature 437(7057):376–380.
2009	MIQE guidelines first published Established guidelines for conducting and reporting PCR to help enable PCR reliability and reproducibility between labs.	Bustin SA, Benes V, Garson JA, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.Clin Chem 55(4):611–622.

2010s

Year	Milestone	Reference
2011	dPCR commercialized First dPCR instrument available in market.	Butkus B (2010) Digital PCR Space Heating up as Life Science Tool Vendors Begin Staking Claims.genomeweb
2012	CRISPR-Cas9 discovered Demonstrated the ability to program the Cas9 protein with guide RNAs to target specific DNA sequences for cleavage, marking the development of CRISPR-Cas9 as a genome-editing tool.	Jinek M, Chylinski K, Fonfara I, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337(6096):816–821.
2016	Digital PCR demonstrated for low-abundance targets Highlights the advantages of digital PCR by pairing the QuantStudio 3D Digital PCR System and ProFlex Thermal Cycler; digital PCR improves mitochondrial DNA deletion detection.	Belmonte FR, Martin JL, Frescura K et al. (2016) Digital PCR methods to improve detection sensitivity and measurement precisions of low-abundance mtDNA deletions. Sci Rep 6:25186.

2020s

Year	Milestone	Reference
2020	PCR becomes a household term COVID pandemic brings PCR into the public vernacular and household discussions.	Velavan TP, Meyer CG (2020) COVID-19: A PCR-defined pandemic.Int J Infect Dis 103:278–279.
2022	cfDNA as a non-invasive biomarker Digital PCR provides sensitivity and specificity to use cell-free DNA as a non-invasive biomarker.	Edwards RL, Menteer J, Lestz RM, et al. (2022) Cell-free DNA as a solid-organ transplant biomarker: technologies and approaches.Biomark Med 16(5):401–415.
2024	Implications of sex in disease explored Loss of Y chromosome on cell studied to help understand the implications of sex related to disease.	Celli L, Gasparini P, Biino G et al. (2024) CRISPR/Cas9 mediated Y-chromosome elimination affects human cells transcriptome.Cell Biosci 14(1):15.
2024	Advances in prime editing Prime editing, using a reverse transcriptase enzyme, advances to improve cellular gene editing capabilities and accuracy.	Yan J, Oyler-Castrillo P, Ravisankar P et al. (2024) Improving prime editing with an endogenous small RNA-binding protein. Nature 628:639–647.