Polymerase chain reaction: Difference between revisions

Latest revision as of 01:15, 20 February 2025

Polymerase Chain Reaction (PCR) is a widely used laboratory technique in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.

Overview[edit]

PCR enables the selective amplification of a specific region of DNA from a complex pool of DNA. The process relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repetitive amplification.

Process[edit]

The PCR process generally involves several key steps:

Denaturation: The double-stranded DNA is heated to a high temperature to separate it into two single strands.
Annealing: Temperature is lowered to enable the DNA primers to attach to the template DNA.
Extension: DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs (deoxynucleoside triphosphates) in a sequence dictated by the DNA template and primers.

This cycle is typically repeated 25-35 times to produce the required quantity of DNA.

Applications[edit]

PCR has a broad range of applications including:

Genetic testing: Identifying genetic disorders from small samples of DNA.
Forensic science: Amplifying DNA from crime scene evidence.
Infectious disease: Detecting the presence of pathogen DNA in blood or tissues.
Research: Cloning DNA sequences and generating probes for Southern blotting.

Variations[edit]

Several variations of PCR exist, including:

Real-Time PCR or Quantitative PCR (qPCR): Allows quantitative measurement of DNA.
Reverse Transcription PCR (RT-PCR): Used for amplifying DNA from RNA.
Multiplex PCR: Allows amplification of multiple targets in a single PCR setup.

Challenges and Limitations[edit]

While PCR is a powerful and versatile tool, it is not without limitations. These include the potential for contamination leading to false results, the need for precise thermal cycling, and limitations in the size of DNA that can be amplified.

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-The '''Polymerase Chain Reaction''' ('''PCR''') is a biochemical technology in molecular biology that amplifies a single or few copies of a piece of [[DNA]] across several orders of magnitude, generating thousands to millions of copies of a particular [[DNA sequence]].<ref name="saiki1988">{{cite journal | last1 = Saiki | first1 = R. K. | last2 = Gelfand | first2 = D. H. | last3 = Stoffel | first3 = S. | last4 = Scharf | first4 = S. J. | last5 = Higuchi | first5 = R. | last6 = Horn | first6 = G. T. | last7 = Mullis | first7 = K. B. | last8 = Erlich | first8 = H. A. | title = Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase | journal = Science | volume = 239 | issue = 4839 | pages = 487–491 | year = 1988 | pmid = 2448875 | doi = 10.1126/science.2448875}}</ref>
+'''Polymerase Chain Reaction''' (PCR) is a widely used [[laboratory]] technique in [[molecular biology]] to amplify a single copy or a few copies of a segment of [[DNA]] across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by [[Kary Mullis]], PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.
-[[File:COLD-PCR applications.jpg|thumb|COLD-PCR applications]]
-=== Procedure ===
-The PCR technique is used in molecular biology to make several copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications. These include [[DNA cloning]] for sequencing, DNA-based [[phylogeny]], or functional analysis of [[gene]]s; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.<ref name="bustin2004">{{cite book | last1 = Bustin | first1 = S. A. | title = A–Z of Quantitative PCR | publisher = International University Line | year = 2004 | isbn = 978-0-9636817-8-6}}</ref>
+==Overview==
-[[File:Polymerase chain reaction diagram.jpg|thumb|Polymerase chain reaction diagram]]
+PCR enables the selective amplification of a specific region of DNA from a complex pool of DNA. The process relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repetitive amplification.
-PCR follows three steps, which are repeated for 20 to 40 cycles. This cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage.
-* '''Denaturation''' at 94–96°C: During the denaturation, the double strand melts open to single-stranded DNA, all enzymatic reactions stop (for example, the extension from a previous cycle).
-* '''Annealing''' at ~68°C: The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single-stranded primer and the single-stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double-stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
-* '''Elongation''' at 72°C: The DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand.<ref name="innis1990">{{cite book | last1 = Innis | first1 = M. A. | last2 = Gelfand | first2 = D. H. | title = PCR Protocols | publisher = Academic Press | year = 1990 | isbn = 978-0-12-372180-8}}</ref>
-=== Variations of PCR ===
+==Process==
+The PCR process generally involves several key steps:
+# '''Denaturation''': The double-stranded DNA is heated to a high temperature to separate it into two single strands.
+# '''Annealing''': Temperature is lowered to enable the DNA primers to attach to the template DNA.
+# '''Extension''': DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs (deoxynucleoside triphosphates) in a sequence dictated by the DNA template and primers.
-Several variations of PCR have been developed for different applications.
+This cycle is typically repeated 25-35 times to produce the required quantity of DNA.
-* '''Reverse transcription PCR (RT-PCR)''': This is used for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified.<ref name="bustin2004"/>
+==Applications==
-* '''Real-time PCR''': Also known as quantitative PCR (qPCR), it monitors the amplification of a targeted DNA molecule during the PCR, i.e., in real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively, such as in a comparative [[Cq (quantification cycle)]] value relative to the amount of DNA input or using absolute quantification.
+PCR has a broad range of applications including:
-* '''Multiplex-PCR''': Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform.<ref name="mackay2002">{{cite journal | last1 = Mackay | first1 = I. M. | title = Real-time PCR in the microbiology laboratory | journal = Clinical Microbiology and Infection | volume = 10 | issue = 3 | pages = 190–212 | year = 2004 | pmid = 15008936 | doi = 10.1111/j.1198-743x.2004.00822.x}}</ref>
+* [[Genetic testing]]: Identifying genetic disorders from small samples of DNA.
-* '''Hot-start PCR''': A technique performed manually by heating the reaction components to the DNA melting temperature (e.g., 95°C) before adding the polymerase.<ref name="innis1990"/>
+* [[Forensic science]]: Amplifying DNA from crime scene evidence.
-* '''Assembly PCR''': Assembly PCR or Polymerase Cycling Assembly (PCA) is the synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.
+* [[Infectious disease]]: Detecting the presence of pathogen DNA in blood or tissues.
+* [[Research]]: Cloning DNA sequences and generating probes for Southern blotting.
-=== Applications of PCR ===
+==Variations==
+Several variations of PCR exist, including:
+* '''Real-Time PCR''' or Quantitative PCR (qPCR): Allows quantitative measurement of DNA.
+* '''Reverse Transcription PCR''' (RT-PCR): Used for amplifying DNA from [[RNA]].
+* '''Multiplex PCR''': Allows amplification of multiple targets in a single PCR setup.
-PCR has a wide range of applications in different areas of biology and medicine, including:
+==Challenges and Limitations==
+While PCR is a powerful and versatile tool, it is not without limitations. These include the potential for contamination leading to false results, the need for precise thermal cycling, and limitations in the size of DNA that can be amplified.
-* '''Molecular biology''': PCR is the cornerstone of modern molecular biology.
+==See Also==
-* '''Medical and diagnostic applications''': PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer diagnosis and in forensic medicine.
-* '''Genetic testing''': PCR is used to analyze genes associated with genetic disorders, including [[polymerase chain reaction#Disease-causing mutations|mutations in oncogenes and tumor suppressor genes in cancer]].
-* '''Infectious diseases''': PCR allows for the identification of infectious diseases, such as HIV, HPV, Chlamydia trachomatis, and Neisseria gonorrhoeae.
-* '''Forensic sciences''': PCR is used in forensic sciences to identify individuals or animals by their DNA profiles.<ref name="bustin2004"/>
-== References ==
-{{reflist}}
-== See also ==
 * [[DNA sequencing]]
-* [[Real-time polymerase chain reaction]]
+* [[Molecular cloning]]
-* [[Reverse transcription polymerase chain reaction]]
+* [[Gene expression]]
-* [[Taq polymerase]]
-[[Category:Molecular biology techniques]]
-[[Category:DNA replication]]
-[[Category:Genetic engineering]]
-[[Category:Laboratory techniques]]
-[[Category:Polymerase chain reaction]]
-== External links ==
-[[National Institutes of Health]]: [https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction Polymerase Chain Reaction (PCR)]
+[[Category:Molecular biology]]
-[https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr Khan Academy: Polymerase Chain Reaction (PCR)]
+[[Category:Biotechnology]]
-== Further Reading ==
+[[Category:DNA]]
+[[Category:Genetics]]
-[https://pubmed.ncbi.nlm.nih.gov/2448875/ Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., ... & Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. ''Science, 239''(4839), 487-491.]
+{{medicine-stub}}
-[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878667/ Bustin, S. A., & Nolan, T. (2017). Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research. ''European Journal of Clinical Investigation, 47''(10), 756-774.]
+<gallery>
-{{DEFAULTSORT:Polymerase Chain Reaction}}
+File:PCR tubes.png|Polymerase chain reaction
-[[de:Polymerase-Kettenreaktion]]
+File:PCR masina kasutamine.jpg|Polymerase chain reaction
-[[fr:Réaction en chaîne par polymérase]]
+File:Primitive PCR machine for scrap.JPG|Polymerase chain reaction
-[[it:Reazione a catena della polimerasi]]
+File:Polymerase chain reaction-en.svg|Polymerase chain reaction
-{{stub}}
+File:Roland Gel.JPG|Polymerase chain reaction
+File:Tucker PCR.png|Polymerase chain reaction
+File:Exponential Amplification.svg|Polymerase chain reaction
+File:Pcr fingerprint.png|Polymerase chain reaction
+File:US Army CID agents at crime scene.jpg|Polymerase chain reaction
+File:Primers RevComp.svg|Polymerase chain reaction
+File:Baby Blue - a prototype polymerase chain reaction (PCR), c 1986. (9663810586).jpg|Polymerase chain reaction
+</gallery>