What Is Polymerase Chain Reaction? PCR History, Procedure and Medical Importance
In this article
Polymerase chain reaction (PCR) is a method that makes millions of copies of a DNA fragment in the laboratory. Conceived during a nighttime journey in 1983, within forty years it became a foundation for genetic diagnosis, cancer treatment selection, forensic medicine and the testing infrastructure of a global pandemic. This article explains PCR from the beginning and corrects several widespread misconceptions, citing their sources.
The simplest analogy for understanding PCR is this: you have a single page, too faint to read. PCR makes it readable by photocopying it billions of times. But there is an important difference — a photocopier copies the entire page, whereas PCR copies only the paragraph you have marked . The marking is performed by primers , short synthetic DNA fragments. This selectivity transforms the method from a copying tool into a diagnostic tool in practice.
In oncology, PCR is not an abstract laboratory technique. The 21-gene test that determines whether chemotherapy is needed in breast cancer is an RT-PCR test. Treatment response in chronic myeloid leukemia is measured using PCR. The HPV test used for cervical cancer screening is PCR-based. The EGFR test that determines which targeted drug should be used in lung cancer is PCR. Thus, in oncology, PCR is not “background technology” but the treatment decision itself.
What Is PCR in One Sentence? Exponential amplification of a DNA sequence bounded at both ends by synthetic primers, using heating–cooling cycles and a heat-resistant DNA polymerase enzyme. The copy number theoretically doubles each cycle; after 30 cycles, a single molecule produces approximately one billion copies.
How Does PCR Work? Three Steps
PCR consists entirely of changing temperature in a specific sequence. A tube contains target DNA, two primers, DNA polymerase, four nucleotides (A, T, G, C) and a suitable buffer solution. The instrument — a thermal cycler — changes the tube's temperature automatically.
| Step | Temperature | Duration | What happens? |
|---|---|---|---|
| 1. Denaturation | 94–98 °C | 20–30 seconds | Hydrogen bonds holding the double helix together break; the two strands separate. |
| 2. Primer binding (annealing) |
50–65 °C | 20–40 seconds | Synthetic primers attach to both ends of the target region, defining the area to be amplified. |
| 3. Elongation (extension) |
72 °C | Varies with length | DNA polymerase synthesizes the new strand, starting at the end of the primer. |
These three steps are repeated 25–40 times. The copy number theoretically doubles with each cycle.
A correction: the annealing temperature is not “37–65 °C.” Online and in many Turkish sources, the primer annealing temperature is given as a range starting at 37 °C. This information belongs to PCR's early years, the 1983–1985 period when the heat-sensitive Klenow enzyme was used . In modern PCR, the annealing temperature is set approximately 3–5 °C below the primers' melting temperature (Tm) ; typical primers of 18–25 nucleotides have a Tm of 55–65 °C, so the practical range is 50–65 °C. Working at 37 °C causes primers to attach in the wrong places, producing nonspecific products. This difference is not academic: in a diagnostic test, it means a false positive.
Why Is a Heat-Resistant Enzyme Essential?
Early PCR had a major practical problem. Each cycle heated the tube to 94 °C to separate DNA; this separated DNA but also destroyed the enzyme. The tube therefore had to be opened and fresh enzyme added manually at the start of every cycle. A 30-cycle experiment meant 30 manual enzyme additions — automation was impossible.
The solution came from a bacterium living in hot springs. Thermus aquaticushas a DNA polymerase — abbreviated Taq polymerase — works best at 75–80 °C and even at 95 °C has a half-life of approximately 40 minutes. Once Taq entered PCR, opening the tube was no longer necessary, and the process became fully automated. This step transformed PCR from a laboratory trick into an industrial standard in practice.
Taq has one weakness: it lacks 3'→5' proofreading activity, meaning it cannot go back and correct a copying error. The reported error rate of the original enzyme is approximately one in 9.000 nucleotides. This is not a problem for most diagnostic applications, but for sequencing or cloning, where a single letter matters, other heat-resistant polymerases with proofreading activity, such as Pfu or Phusion, are preferred. Saying “PCR was performed” does not mean a single method using a single enzyme was applied.
PCR's History — and Eight Widespread Errors
PCR's story is mostly told correctly in Turkish sources, but several dates and a myth are repeated continually. The following table places statements widespread online and included in this article's previous version alongside verified corrections.
| Circulating statement | The correct information |
|---|---|
| “Presented in 1985 by Erlich, Mullis and Saiki” | The paper has seven authors, and the first author is Saiki: Saiki, Scharf, Faloona, Mullis, Horn, Erlich, Arnheim. Science 1985;230(4732):1350–1354. |
| “In 1966, Nirenberg, Khorana and Holley deciphered the genetic code and synthesized an artificial gene; Khorana received the Nobel Prize for this” | The Nobel Prize was awarded in 1968for “interpreting the genetic code and its function in protein synthesis”— not an artificial gene. Khorana's first complete gene synthesis was in 1972, four years after the Nobel Prize. |
| “Taq polymerase, discovered in 1976, was obtained from Thermus aquaticus” | Two separate events have been combined. The bacterium was discovered in 1969 by Thomas Brock and Hudson Freeze at Yellowstone. The enzyme was purified in 1976 by Chien, Edgar and Trela (J Bacteriol 1976;127:1550–1557). |
| “An enzyme resistant to 94 °C” | Correct but incomplete: Taq's optimum operating temperature is 75–80 °C; at 95 °C, its half-life is approximately 40 minutesand at 92,5 °C, more than 2 hours. |
| “The paper was rejected by about 15 journals” | Unsupported. Two rejections are documented: Nature and Science. In his Nobel lecture, Mullis personally describes the Science rejection. The methods paper was published in 1987 in Methods in Enzymology. No reliable source could be found for “15 journals.” |
| “Taq polymerase was added to PCR in 1986” | The paper describing PCR with Taq dates to 1988 : Saiki et al., Science 1988;239(4839):487–491. |
| “A year later, Perkin Elmer developed the thermal cycler” | The order has been reversed. Cetus's prototype instrument, “Mr. Cycle,” was built in 1985–86; the first commercial instrument was Perkin-Elmer Cetus's TC1 DNA Thermal Cycler, 1987. Thus, the instrument came before the Taq paper. |
| “Primer annealing at 37–65 °C” | In modern PCR, 50–65 °C; 3–5 °C below primer Tm. The 37 °C temperature belongs to the 1983–85 Klenow enzyme period. |
The fifth of these corrections is the most instructive. The story “the genius's paper was rejected by 15 journals” is a much-loved science communication trope: the system was blind, the individual right. The real story is more interesting and realistic — two major journals rejected it, the method appeared in a methods journal, and the 1985 application paper that provided the actual breakthrough had already appeared in Scienceat that time. The system was not blind; the idea itself was unconvincing until it was shown to work. This is a much more accurate account of how science works than the “15 rejections” myth.
The Night the Idea Arrived
According to Mullis's own account, the idea came on a Friday night in spring 1983 while driving from Berkeley to his cabin in northern California, on Highway 128 . He pulled over, took paper from the glove compartment and wrote it down. His Nobel biography describes the moment as “when the California buckeyes were in bloom.” The first successful demonstration of the method is recorded as 16 December 1983 in the account.
The idea's essence was simple, perhaps exactly why nobody had thought of it: DNA polymerase was already known (1956), primers could already be synthesized, and heat separation of DNA was already known. Mullis did not discover a new molecule but put three known things into a cycle. Exponential amplification emerged from this.
Its First Published Application Was a Diagnostic Test. The 1985 Science paper concerned not basic biology but the diagnosis of sickle cell anemia: amplification of the β-globin gene followed by restriction analysis. PCR was introduced to the world not as a research tool but directly as a clinical diagnostic tool . Forty years later, considering its place in oncology, this beginning seems meaningful.
Kary Mullis: Separating the Method from the Person
Mullis shared the 1993 Nobel Prize in Chemistry with Michael Smith ; Smith received it for work on site-directed mutagenesis. Mullis's citation was a single sentence: “for his invention of the polymerase chain reaction method.”
Mullis's later years, however, are among the uncomfortable examples in scientific history. He is documented as having denied that HIV causes AIDS — despite having no research background in either field. He downplayed human-caused climate change and ozone depletion and alleged scientists colluded for funding. He professed belief in astrology and described in his autobiography encountering a glowing, talking raccoon that he thought might be extraterrestrial. He died on 7 August 2019, aged 74, from pneumonia complications.
Writing this is necessary because the opposite reasoning is widespread. The statement “the man who invented PCR denied the HIV–AIDS link” was repeatedly used during the pandemic to discredit PCR tests. The reasoning is flawed in both directions: Winning a Nobel Prize does not make someone right about everything; being wrong about other issues does not invalidate their method. PCR is valid independently of Mullis's opinions because it has produced reproducible results in millions of experiments over forty years. A method stands not on its inventor's authority but on its reproducibility . This distinction also filters claims confronting cancer patients, such as “that famous scientist said this.”
PCR Is Not One Method: Four Generations
PCR in the 1980s was not the same as PCR in an oncology laboratory today. The method evolved through four generations, each created to answer a question its predecessor could not.
| Generation | Year | Question answered | Measurement method |
|---|---|---|---|
| Conventional (endpoint) PCR | 1983–85 | “Is this sequence present or absent?” | After cycling, a gel is checked for a band — qualitative |
| RT-PCR (reverse transcription) |
Late 1980s | “Is this RNA present? How much is this gene expressed?” | RNA is first converted to cDNA, then amplified |
| Real-time PCR (qPCR) | 1992 | “How much is present?” | Fluorescence is measured every cycle — relative quantitative |
| Digital PCR (dPCR/ddPCR) | 1999 (concept) | “How many molecules are present? And among 100.000 normal molecules, can you find 1 mutant?” | The sample is divided into thousands of droplets, and positive droplets are counted — absolute quantitative |
Real-Time PCR (qPCR) and the Ct Value
Conventional PCR had this problem: measurements were made after the reaction ended, when materials in the tube were depleted and amplification had plateaued. A sample initially containing 10 copies and one containing 10.000 produced similar final bands. Thus, you could answer “is it present?” but could not answer “how much is present?” as a quantitative question.
The study published by Higuchi and colleagues in 1992 changed this: observing amplification not after it ended but while it happened . A fluorescent system is added to the tube, and the instrument measures light every cycle. In widely used TaqMan chemistry, a probe binds to the target; one end carries a light-emitting molecule and the other a quencher suppressing it. During extension, Taq polymerase encounters and breaks the probe, separating the ends and releasing light. The light increase is proportional to amplification.
What Is Ct (or Cq)? It is the cycle number at which fluorescence crosses the threshold (threshold cycle / quantification cycle; the MIQE guideline prefers “Cq”). The logic is reversed and often confused: The lower the Ct, the higher the initial target amount. A reaction starting with many copies crosses the threshold earlier. This explains pandemic debates such as “my Ct is 32; does that mean little virus?” — most overlooked that Ct is a relative number varying by instrument and protocol rather than an absolute measure.
Digital PCR: Measuring by Counting
qPCR measures relatively — saying “this many times more” than a reference. Digital PCR uses different logic: it divides a sample into thousands of small compartments or droplets, each containing either one target molecule or none. After cycling, it countshow many compartments are positive. Poisson statistics directly calculate the initial molecule count — without a standard curve.
The concept was named in 1999 by Bert Vogelstein and Kenneth Kinzler , and their first demonstrated application directly concerned this article's subject: detecting rare cancer mutations. There were predecessors — Cetus researchers demonstrated detection of a single β-globin copy through dilution in 1988, and Morley and Sykes made limiting dilution a quantitative technique in 1992.
Digital PCR's importance in oncology comes from one feature: finding what is rare. Reported performance is as follows — mutant DNA can be detected among a 200.000-fold excess of normal DNA ; compared with conventional qPCR it is reportedly approximately 2.000 times more sensitive; one in 1.250.000 cells is the rarity of sequences that can be detected. Because circulating tumor DNA (ctDNA) often accounts for less than one-thousandth of all cell-free DNA, this sensitivity is a prerequisite, not a luxury. The first droplet digital PCR system approved for clinical use received CE marking in 2017 and FDA approval for chronic myeloid leukemia in 2019 for clinical use.
RT-PCR and the Pandemic
RNA cannot be amplified directly; it must first be converted to DNA (cDNA) by reverse transcriptase. The method incorporating this step is called RT-PCR . Because SARS-CoV-2 is an RNA virus, its test followed this logic: reverse transcription followed by real-time amplification — RT-qPCR. Its ability to detect and quantify in a single closed run made it the reference test.
The pandemic's most lasting lesson about PCR concerns its limits rather than its power. PCR finds the target sequence — it does not say whether that sequence belongs to a living, infectious organism . Fragments of viral RNA detectable for weeks after infection resolved could therefore produce positive results. The same limitation applies in oncology and receives less discussion: PCR shows the presence of a mutation, not whether the cell carrying it is growing the tumor. Using the method correctly requires knowing both what it says and what it does not say as part of its interpretation. A reliable figure for the total number of tests performed worldwide during the pandemic could not be verified for this article; therefore, none is given.
PCR in Oncology: Four Concrete Examples
PCR's role in cancer medicine goes far beyond “used in research.” The following four examples are situations where a PCR result directly becomes a treatment decision in clinical practice.
1. Chemotherapy Decisions in Breast Cancer: Oncotype DX
In hormone receptor-positive, HER2-negative early breast cancer, a difficult question is whether to give chemotherapy after surgery. The Oncotype DX Breast Recurrence Score test addresses this through tumor gene expression. According to the manufacturer, it evaluates the expression of 21 genes — 16 cancer-related genes and 5 reference genes — using “reverse transcription polymerase chain reaction (RT-PCR) to analyze tumor RNA” . The sample is tissue taken during core needle biopsy or surgical excision.
| Study | Patients | Population | Result |
|---|---|---|---|
| TAILORx | 10.273 | Node-negative; those scoring 11–25 were randomized | Invasive disease-free survival at 9 years %83,3 (endocrine therapy alone) — %84,3 (endocrine + chemotherapy); overall survival %93,9 – %93,8. In other words, no benefit from chemotherapy. Exception: a small benefit was seen in those aged 50 or younger with scores 16–25. |
| RxPONDER | 5.083 | 1–3 positive lymph nodes, score ≤25 | Invasive disease-free survival at 5 years — postmenopausal: %91,9 versus %91,3 (no difference); premenopausal: %89,0 versus %93,9 (favoring chemotherapy). |
Sources: NCI press release (TAILORx) and SWOG announcement (RxPONDER).
The real news here is not which patients the test assigns chemotherapy to, but which it does not. . TAILORx allowed tens of thousands of node-negative women with intermediate scores to avoid chemotherapy and its permanent side effects — through an RT-PCR test. RxPONDER showed that results reversed by menopausal status, demonstrating that one number does not mean the same thing for every patient. We discussed the clinical details of this decision in patients who will not receive chemotherapy after breast cancer surgery and compared genomic tests in genomic recurrence score tests in lobular breast cancer in our related articles.
2. Measuring Response in Chronic Myeloid Leukemia: BCR-ABL1
Chronic myeloid leukemia is the disease in which PCR is most deeply established in oncology. Its driver, BCR-ABL1 , is a fusion transcript measured by quantitative RT-PCR, with results reported on the International Scale (IS) . Here, treatment success is defined not by “has the tumor shrunk?” but by how many logarithms a number has fallen in its measurement.
| Level | Definition | Meaning |
|---|---|---|
| %1 IS | BCR-ABL1 ≤ %1 | Equivalent to complete cytogenetic response |
| MMR / MR3 | BCR-ABL1 ≤ %0,1 | Compared with baseline: 3 log reduction — major molecular response |
| MR4 | ≤ %0,01 or undetectable with >10.000 ABL1 transcripts | 4 log reduction |
| MR4.5 | ≤ %0,0032 or undetectable with >32.000 ABL1 transcripts | 4,5 log reduction |
| MR5 | ≤ %0,001, with >100.000 ABL1 transcripts | 5 log reduction |
Response milestones are generally assessed at months 3, 6 and 12 . Transcript counts in these definitions are sensitivity requirements: reporting MR4.5 requires the laboratory to have amplified at least 32.000 control transcripts.
This table is the most striking indicator of PCR's sensitivity. Reliably measuring a proportion of three in ten thousand means counting a few molecules per million — routinely performed every three months in a hospital laboratory. A deep, sustained molecular response permits consideration of stopping the tyrosine kinase inhibitor in some patients, a trial of treatment-free remission. The response depth and duration required for a discontinuation trial could not be verified for this article; this decision belongs to the hematologist, and no numerical threshold is given here.
3. HPV Testing in Cervical Cancer Screening
Over the past twenty years, cervical cancer screening has shifted from examining cells (cytology/smear) to examining viral nucleic acid . The American Cancer Society's 2020 guideline explicitly recommends “primary HPV testing (HPV testing without a Pap test) every five years from age 25 to 65 as the preferred method” ; co-testing and cytology alone remain “acceptable alternatives” because access to HPV tests approved for primary screening may not be universally available.
A distinction is necessary here: not all HPV tests are PCR. The Aptima HPV test detects not viral DNA but E6/E7 mRNA and uses different amplification chemistry. This distinction matters — detecting mRNA demonstrates not simply the virus's presence but that its oncogenes are active as its intended indication.
New development: self-collection. The FDA, on 14 May 2024, approved self-collection for two HPV tests — BD Onclarity HPV and Roche cobas HPV — allowing women to collect their own samples. Approval covers collection “in a healthcare setting, such as primary care offices, urgent care centers, pharmacies and mobile clinics” . This is an important milestone for women who never attend screening because they hesitate to undergo a gynecological examination. We discussed cervical cancer prevention through screening in detail in our cervical cancer article previously.
4. Predicting Treatment Response in Glioblastoma: MGMT Methylation
Whether the MGMT gene promoter is methylated predicts the likelihood of benefiting from temozolomide chemotherapy in glioblastoma. The main method of determining this is methylation-specific PCR (MSP); the second is pyrosequencing (PSQ).
Neuro-Oncology published a Cochrane-based meta-analysis quantifying how dominant the method is: among approximately 190 studies using a single technique (27.710 participants), 94 studies used MSP and 27 used pyrosequencing . Comparing predictive power in glioblastoma patients receiving temozolomide, relative to immunohistochemistry, MSP's ratio of hazard ratios (RHR) was 1,31 (%95 CI 1,01–1,71)and pyrosequencing's was 1,36 (1,01–1,84) ; the difference between pyrosequencing and MSP was not significant (1,14; 0,87–1,48). Regarding thresholds, for CpG regions 74–78, a %9threshold reportedly performed better than higher thresholds of %28–29.
This last sentence reveals a rarely discussed aspect of molecular tests: two laboratories examining the same sample with the same method but different thresholds may give the same patient different results. Saying “MGMT methylated” is incomplete without knowing the method and threshold used. We discussed how molecular diagnosis determines glioblastoma treatment in molecular diagnosis of glioblastoma and IDH-wildtype high-grade glioma in our related articles.
PCR-Based Companion Diagnostics
The FDA's official companion diagnostic list includes many PCR tests named in drug labels that determine who should receive those drugs. The following entries come directly from this list.
| Test | Manufacturer | PMA no. | Associated drug(s) |
|---|---|---|---|
| cobas EGFR Mutation Test v2 | Roche Molecular Systems | P120019 | Osimertinib, erlotinib, gefitinib, afatinib |
| therascreen KRAS RGQ PCR Kit | QIAGEN | P140022 | Cetuximab, panitumumab |
| cobas KRAS Mutation Test | Roche Molecular Systems | P140023 | Cetuximab, panitumumab |
| therascreen PIK3CA RGQ PCR Kit | QIAGEN | P180001 | Alpelisib |
| cobas 4800 BRAF V600 Mutation Test | Roche Molecular Systems | P110020 | Vemurafenib; later cobimetinib |
| therascreen BRAF | QIAGEN | P130010 | Vemurafenib |
Manufacturer, PMA number and associated drug information are taken from the FDA companion diagnostic list. Approval years were not individually verified for this article and are therefore omitted from the table — each test's date can be checked using its PMA number at accessdata.fda.gov.
This table summarizes PCR's role in oncology at a glance: in lung cancer which patient receives an EGFR inhibitor, in colorectal cancer which patient should not receive an anti-EGFR antibody, and in melanoma which patient is eligible for a BRAF inhibitor — all depend on a PCR result. The type of carcinoma is identified by pathology, but the pathology report alone is no longer sufficient; a molecular report accompanies it.
Liquid Biopsy and Minimal Residual Disease: Is PCR's Era Ending?
Determining whether microscopic disease remains after surgery by measuring circulating tumor DNA (ctDNA) — that is, minimal residual disease (MRD) monitoring — is among oncology's fastest-growing fields. Given digital PCR's sensitivity for rare variants, one might expect this to be PCR's natural domain. The reality is more complicated.
A common misconception needs correcting. Signatera, a commonly used tumor-informed MRD test, is often described as “personalized multiplex PCR.” The manufacturer's current physician page instead describes creating a personalized panel from tumor tissue using whole-exome or whole-genome sequencing to build the panel, 64 clonal variants being selected and sought in blood through “ultra-deep sequencing at up to 350.000-fold depth for each selected variant” . Thus, the readout step is sequencing, not PCR. The same page explicitly states that the test is not FDA-approved (“have not been cleared or approved by the US Food and Drug Administration”); the laboratory operates with CLIA and CAP accreditation, and Medicare covers the test for monitoring disease progression, recurrence and relapse.
This does not mean PCR is declining, but that the division of labor is changing. When you do not know which mutations are in the tumor, sequencing is needed — PCR only finds what you seek. But when you know which mutation to seek, PCR is faster, cheaper and often sufficiently sensitive. We discussed the clinical implications of ctDNA-guided treatment changes in the GALAXY study and SERENA-6: treatment switching according to ESR1 mutation in our related articles; blood-test screening is covered in colon cancer screening based on fragmentomics and methylation in our related articles.
PCR or Next-Generation Sequencing?
This question is frequently asked and usually framed incorrectly. The two are not competitors but tools for different questions. In the National Cancer Institute's words, some biomarker tests “look for a single biomarker,” while others “look for many biomarkers at once and may be called multigene or panel tests”; others read the whole exome or whole genome.
| PCR-based test | Next-generation sequencing (NGS) | |
|---|---|---|
| Basic logic | Finds what you seek | Reads what is present |
| Target | A limited number of predefined variants | Broad panel, exome or genome |
| Unknown variant | Cannot detect it — no primer was designed | Can detect it |
| Regulatory status | May be a companion diagnostic named in a drug label | Broad-panel approvals also exist |
| Typical use | Making a single decision | Mapping the landscape |
Note: This table deliberately omits comparisons of turnaround time, cost and sensitivity. No authoritative source for widely circulating figures on these topics could be verified while preparing this article; the fields were left empty rather than estimated.
The structural argument is: The FDA continues to approve single-variant PCR tests as companion diagnostics today, and their names appear in drug labels. If sequencing had completely replaced PCR, the PCR entries in this list would disappear. They have not. Sequencing maps mutations; PCR supports decisions based on a known mutation — the two work together. Why a mutation's location within a gene matters is shown by studies that are themselves products of precisely this two-layer approach.
PCR's Limitations and Sources of Error
No method can be used correctly without understanding its limitations. PCR's most frequent clinical problems are:
PCR's strength is its weakness: because even a single molecule can be amplified, airborne product from a previous reaction can cause a false positive. Molecular laboratories therefore require separate rooms and unidirectional workflows.
Blood hemoglobin, residual formalin in tissue or heparin may inhibit the enzyme, producing a false negative — undetected unless an internal control is included.
PCR sees only the sequence for which primers were designed. A mutation outside the panel is reported as “no mutation.” This means a negative result must always be interpreted carefully.
Taq lacks proofreading activity and makes approximately one error per 9.000 nucleotides. This is unimportant in diagnostic tests but important when amplifying before sequencing.
DNA is fragmented in paraffin-embedded tissue. Long targets do not amplify. Test failure is often a problem with the sample rather than the method.
PCR finds a sequence, not whether the organism carrying it is alive or the cell is active. Interpretation always includes clinical context.
Applications of PCR
| Field | Application |
|---|---|
| Oncology | Companion diagnostics (EGFR, KRAS, BRAF, PIK3CA) |
| Gene expression-based recurrence scores (21-gene RT-PCR test) | |
| Monitoring molecular response in hematological malignancies (BCR-ABL1) | |
| Determining methylation status (MGMT) | |
| Genetics | Carrier testing and diagnosis of inherited diseases |
| Prenatal diagnosis | |
| Preimplantation genetic testing in IVF | |
| Infection | Detecting viral and bacterial pathogens |
| Viral load measurement (HIV, HBV, HCV) | |
| HPV-based cervical cancer screening | |
| Forensic medicine | Identification and paternity testing |
| DNA profiling from very small samples | |
| Research | Cloning, amplification before sequencing |
| Examining DNA–protein interactions | |
| Evolutionary biology and ancient DNA studies | |
| Food and industry | Species identification and adulteration testing |
| Allergen and genetically modified organism detection |
The Situation in Türkiye
HPV Testing in the National Screening Program
Türkiye's National Standards for the Cervical Cancer Screening Program define screening as: “population-based screening for women starting at age 30 and ending at age 65” and “the ideal method is screening with an HPV or Pap-smear test every five years”. The statement “HPV or Pap-smear testing is repeated every five years” establishes the five-year interval.
Sample collection is performed by “healthcare personnel trained in this field working at an ASM or TSM (KETEM)” ; collected samples are sent “under appropriate fixation conditions for examination to centers designated by the Public Health Institution of Türkiye or Public Health Directorates” . The program therefore relies on centralized laboratories — the technical prerequisite for population-based HPV testing.
The current document version, present program operation, test brands and coverage rates could not be verified for this article. The available text is the official document defining program standards; current implementation details should be sought from the Ministry of Health's own sources. For an overview of the cancer burden in our country, consult Türkiye cancer statistics in our related article.
Molecular Pathology and Reimbursement
This article makes no claims regarding SGK reimbursement. Whether gene expression-based recurrence score tests such as Oncotype DX, PCR-based companion diagnostics or ctDNA/MRD tests are reimbursed in Türkiye could not be verifiedfor this article. Definitive claims circulating on this subject should be treated cautiously; current status should be confirmed with the treating physician and relevant institution. We discussed the overall picture of access to new drugs and tests in the Türkiye access map for new FDA-approved cancer drugs in our related articles.
A practical note: mutation tests such as EGFR, KRAS and BRAF are widely performed in Türkiye at university hospitals, training and research hospitals and private molecular pathology laboratories. For patients, what matters is not only whether the test is performed but also which method and scope are used: a report stating “no KRAS mutation detected” cannot be fully interpreted without knowing which codons the panel includes.
Timeline
DROZDOGAN Academy Commentary
PCR's story is one of the clearest examples of how a good idea spreads through medicine. Yet while updating this article, what stood out was the need to check stories about the method as carefully as the method itself as part of the work.
⚡ Why it matters
Because PCR in oncology is no longer background technology but a decision point. A 21-gene RT-PCR result enables thousands of women to avoid chemotherapy. A BCR-ABL1 measurement determines whether treatment continues. An EGFR test determines which drug is given. For patients, this means: “the test result is not back” means the same as “we cannot start treatment.” Knowing what PCR is helps you understand your own treatment process.
✅ The strength
PCR has endured for forty years not because of its elegance but because of its reproducibility. The same sample, primers and protocol produce the same result anywhere in the world. Reporting a proportion of three in ten thousand on the International Scale in chronic myeloid leukemia concretely demonstrates this standardization: results from laboratories in Ankara and Boston can be compared. Few oncology measurements are standardized this well.
⚠️ The weakness
PCR finds only what you look for . This is the most misleading aspect of negative results: “no mutation detected” often means “none of the mutations in our panel detected,” and these are different. As the MGMT example shows, two laboratories using the same method can use but different thresholds to produce different results for the same patient. Contamination is another issue: a method amplifying a single molecule also amplifies one airborne molecule. PCR's sensitivity is also its vulnerability.
🩺 What to tell patients
(1) When ordering a molecular test, explain that results can take several days to several weeks and that this wait is generally necessary to select the right treatment. (2) Read the report together: show which genes and methods were examined. (3) Explain that a “negative” result is not absolute but depends on the panel's scope. (4) Explain from the start that insufficient tissue may require retesting or a blood sample — one of the most common causes of subsequent delays. (5) Remind patients they will encounter online claims that “PCR tests are unreliable,” mostly originating from issues unrelated to the method.
❓ Open questions
Tumor-informed MRD tests have shifted to sequencing; could digital PCR return as a cheaper, faster alternative? Second: in a ctDNA-positive patient with nothing visible on imaging, does starting treatment early truly extend survival, or do we merely learn of recurrence earlier? This is MRD's central unanswered question. Third: why do gene expression-based recurrence scores reverse with menopausal status — does chemotherapy itself work, or the menopause it induces?
Conclusion: Forty years later, PCR retains its place — now sharing work with sequencing rather than standing alone. As its oncology use expands, how its report is interpreted matters more than the method itself. The most demanding part of updating this article was not adding new information but removing incorrect older information : all eight common statements needed correction. Knowledge is thought not to age; yet the most repeated information becomes outdated fastest.
Frequently Asked Questions
- How Long Does a PCR Test Take?
- The reaction itself generally takes 1–3 hours. A clinical result requires much longer overall: sample collection, transport, DNA/RNA extraction, reaction, interpretation and reporting. Tissue-based molecular oncology tests generally return within days to weeks. This reflects the entire process rather than a slow method.
- What Does “No Mutation Detected” Mean?
- It means the mutations included in the tested panel were not detected — not that no mutations exist. PCR sees only sequences for which primers were designed. The report should state which gene regions were examined; ask if it does not.
- What Is the Difference Between PCR and Next-Generation Sequencing?
- PCR finds what you seek; sequencing reads what is present. PCR suits a decision based on one known mutation, while sequencing maps a tumor's molecular landscape. They complement rather than compete — the FDA still approves single-variant PCR companion diagnostics.
- Does a Low Ct Mean Severe Disease?
- Ct is inversely related to the initial target amount: low Ct means more target. However, Ct is a relative number varying by instrument, kit and sampling technique. Different laboratories' Ct values cannot be compared directly, and Ct alone does not measure disease severity.
- Is Cancer Diagnosed Using PCR?
- No. Cancer is diagnosed through pathology — microscopic tissue examination. After diagnosis, PCR is used to determine the tumor's molecular features , select treatment and monitor response. Screening for precancerous conditions, such as HPV screening, is an exception — even there, definitive diagnosis is still pathological.
- Can a Blood Test Detect Cancer?
- Circulating tumor DNA (ctDNA) can be detected, and the field is developing rapidly. However, current use focuses primarily not on screening but on treatment selection and recurrence monitoring after diagnosis. Many tumor-informed monitoring tests are now sequencing-based rather than PCR, and some operate as laboratory-developed tests without FDA approval.
- Can PCR Give an Incorrect Result?
- Yes. Contamination is the main source of false positives — because PCR can amplify even one molecule, airborne product causes problems. Main false-negative causes are inhibition by blood, formalin or heparin, fragmented sample DNA and the sought variant being absent from the panel. A good test therefore includes an internal control.
- Who Invented PCR? Was It One Person?
- The idea belongs to Kary Mullis, who received the 1993 Nobel Prize in Chemistry for it. However, making the method work, applying it to a disease and enabling automation were achievements of a broad team at Cetus Corporation — the first paper in 1985 had seven authors, with Randall Saiki first.
- Is HPV Screening Performed in Türkiye?
- National standards define population screening for women aged 30–65 using HPV or Pap-smear testing every five years. Trained healthcare personnel collect samples at ASM or KETEM facilities and send them to designated centers. Current operational details and coverage rates could not be verified for this article.
- Are These Tests Reimbursed in Türkiye?
- This article makes no reimbursement claims — SGK coverage of gene expression tests such as Oncotype DX, PCR companion diagnostics and ctDNA tests could not be verified. Current information should be confirmed with the treating physician and relevant institution.
Glossary
- Primer
- A synthetic DNA fragment, usually 18–25 nucleotides long, binding to either end of a target DNA sequence and delimiting the region to be amplified.
- Tm (melting temperature)
- The temperature at which half of a primer separates from its target. Annealing temperature is set 3–5 °C below this.
- Denaturation
- Heat separation of double-stranded DNA into two single strands.
- Taq polymerase
- Thermus aquaticusheat-resistant DNA polymerase obtained from this bacterium. Optimum 75–80 °C; no 3'→5' proofreading activity.
- Thermal cycler
- An instrument changing a PCR tube's temperature in a programmed sequence.
- RT-PCR
- Reverse transcription PCR. RNA is first converted to cDNA, then amplified.
- qPCR / real-time PCR
- PCR in which amplification is monitored by fluorescence every cycle. Produces quantitative results.
- Ct / Cq
- The cycle number at which fluorescence crosses the threshold. A low value means abundant initial target.
- TaqMan probe
- A probe carrying a fluorescent molecule at one end and a quencher at the other. Polymerase cleavage releases light.
- Digital PCR (dPCR/ddPCR)
- A method dividing a sample into thousands of compartments and counting positives. Provides absolute counts without a standard curve.
- Companion diagnostic
- An approved test named in a drug label that determines who should receive the drug.
- ctDNA
- Tumor-derived DNA circulating in blood. Its proportion of all cell-free DNA is often very low.
- Minimal residual disease (MRD)
- Microscopic disease undetectable by imaging after treatment but demonstrable by molecular methods.
- International Scale (IS)
- A standardized reporting scale allowing BCR-ABL1 measurements to be compared between laboratories.
- Methylation-specific PCR (MSP)
- PCR using primers designed to distinguish methylated from unmethylated DNA.
- Contamination
- DNA from earlier reactions or the environment entering a sample. The principal cause of false-positive PCR results.
Transparency note.
Verified: 1985 Science paper's complete author list, volume and pages; Mullis and Faloona's 1987 Methods in Enzymology paper; recipients and citation of the 1968 Nobel Prize in Physiology or Medicine; Khorana's 1972 gene synthesis; Kornberg's 1956 discovery and 1959 Nobel Prize; Thermus aquaticuswas discovered in 1969 by Brock and Freeze at Yellowstone; Taq polymerase was purified in 1976 by Chien, Edgar and Trela, and its optimum temperature; Taq's half-life at 95 °C and error rate; Mullis's own description in his Nobel lecture of the Science rejection; the 1988 Science Taq paper; “Mr. Cycle” and TC1 DNA Thermal Cycler; the sharing and citations of the 1993 Nobel Prize in Chemistry; Mullis's HIV-AIDS, climate and astrology views, death date and age; the 1992 Higuchi study; Vogelstein and Kinzler's introduction of digital PCR in 1999 and reported sensitivity; clinical ddPCR system CE marking in 2017 and FDA approval in 2019; Oncotype DX's 21-gene / 16+5 structure and RT-PCR method (manufacturer page); TAILORx and RxPONDER findings (NCI and SWOG announcements); BCR-ABL1 molecular response definitions; FDA HPV self-collection approval dated 14 May 2024; Aptima as an E6/E7 mRNA test; ACS 2020 screening recommendation; MGMT meta-analysis data; manufacturer, PMA and drug matches in the FDA companion diagnostic list; Signatera's manufacturer-described method and regulatory status; the text of Türkiye's cervical screening standards.
Not verified: Primary sources for deciphering the genetic code (1961, 1966); explicit confirmation of coauthors and source organism in Kornberg's 1956 work; names of engineers who built “Mr. Cycle”; developers of TaqMan chemistry and the first commercial real-time instrument's date; Higuchi's institution in 1992; LAMP's developer and year, so LAMP is omitted; total worldwide pandemic test count; total FDA-approved PCR tests; approval years of companion diagnostics, so absent from the table; response depth and duration required for treatment-discontinuation trials in chronic myeloid leukemia; primary-source confirmation that cobas HPV uses real-time PCR; authoritative turnaround, cost and sensitivity comparisons of PCR and next-generation sequencing; glioblastoma survival figures by MGMT status; current operation, test brands and coverage of Türkiye's cervical screening program; SGK reimbursement status (no claims made).
Nature of the corrections: The eight-item correction table arose from comparing statements in this article's previous version and common Turkish sources against primary and secondary sources. The conclusion that “15 journals rejected it” is unsupported is ours: no reliable supporting source was found, while two rejections (Nature and Science) are documented. The assessment that Mullis's scientific method should be separated from his personal views also belongs to this article.
Declaration of interests: We have no connection with any test, instrument or manufacturer named in this article.
References
- Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. "Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia." Science 1985;230(4732):1350–1354.
- Mullis KB, Faloona FA. "Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction." Methods in Enzymology 1987;155:335–350.
- Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science 1988;239(4839):487–491.
- Chien A, Edgar DB, Trela JM. "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus." Journal of Bacteriology 1976;127(3):1550–1557.
- The Nobel Prize in Chemistry 1993 — Kary B. Mullis and Michael Smith. nobelprize.org
- Mullis KB. Nobel Lecture, 8 December 1993. nobelprize.org
- The Nobel Prize in Physiology or Medicine 1968 — Holley, Khorana, Nirenberg. nobelprize.org
- The Nobel Prize in Physiology or Medicine 1959 — Ochoa, Kornberg. nobelprize.org
- Higuchi R, et al. "Simultaneous amplification and detection of specific DNA sequences." Bio/Technology 1992.
- Vogelstein B, Kinzler KW. Definition of the digital PCR concept, 1999.
- National Cancer Institute. “TAILORx trial: chemotherapy not beneficial for many women with early breast cancer.” Press release, 2018. cancer.gov
- SWOG Cancer Research Network. Announcement of publication of the RxPONDER results in NEJM, 3 December 2021. swog.org
- Exact Sciences. Oncotype DX Breast Recurrence Score — healthcare professional page. exactsciences.com
- U.S. Food and Drug Administration. "List of Cleared or Approved Companion Diagnostic Devices (In Vitro and Imaging Tools)." fda.gov
- National Cancer Institute. "FDA approves self-collection for HPV tests." Cancer Currents Blog, 2024. cancer.gov
- American Cancer Society. Cervical cancer screening guideline, 2020. pressroom.cancer.org
- Hologic. Aptima HPV test product information — E6/E7 mRNA. hologic.com
- Comparative meta-analysis of MGMT promoter methylation methods. Neuro-Oncology 2021;23(9):1457.
- Natera. Signatera — clinician information page. natera.com
- National Cancer Institute. "Biomarker Testing for Cancer Treatment." cancer.gov
- Republic of Türkiye Ministry of Health. National Standards for the Cervical Cancer Screening Program.
- Brock TD, Freeze H. Thermus aquaticusisolation, 1969.
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