Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a technique that amplifies DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure fragments exponentially for analysis. The process is highly specific, allowing for the targeting of specific genomic sequences, even with minuscule sample amounts. The PCR cycles multiple times through 3 phases: denaturation of the template DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure, annealing of a specific primer to the individual DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure strands, and synthesis/elongation of new DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure molecules. From there, the DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure can be visualized with analysis techniques such as gel electrophoresis. The speed, inexpensiveness, ease of use, and high sensitivity and specificity make this technique highly useful for the basic and biomedical sciences, and it has become instrumental in many applications, including forensic analysis, diagnosis of infectious diseases, and diagnosis and screening of genetic abnormalities.

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Editorial responsibility: Stanley Oiseth, Lindsay Jones, Evelin Maza

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Overview

Definition

Polymerase chain reaction (PCR) is a technique that amplifies target DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure sequences, in vitro, exponentially for analysis.

Necessary components

  • Template DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure (sample with the genomic sequence)
  • Primers:
    • Oligonucleotides that are carefully designed for a specific sequence target
    • Complementary to the 3’ end of the target region
  • Free nucleotide bases
  • DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure polymerase:
    • Must be thermostable to survive several rounds of heating/cooling
    • Carries out replication

Procedure

To amplify DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure or RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure, the following stages are repeated in cycles (often up to 30–40 times):

  • Denaturation
  • Annealing of primers
  • Synthesis

Denaturation

  • The sample is heated to approximately 95°C (approximately 203°F).
  • Hydrogen bonds are disrupted → separation of DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure strands
Denaturation

Stage 1: Denaturation—
DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure is denatured at high temperatures, causing separation into single strands.

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Annealing of primers

  • The temperature is reduced to approximately 50–65°C (approximately 122–149°F).
  • Primers bind to the 3’ end of template DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure strands.
  • DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure polymerase binding follows.
Annealing of primers

Stage 2: Annealing of primers—
Primers anneal to the 3’ end of template DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure strands upon cooling of the sample.

Image by Lecturio.

Synthesis/elongation

  • The temperature is increased to approximately 70–72°C (approximatley 158–162°F).
  • DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure polymerase is activated → adds free nucleotides to 3’ end of the primers
  • Creates a new DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure strand identical to the original template
Synthesis elongation

Step 3: Synthesis/elongation—
DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure elongation is catalyzed by a thermostable DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure polymerase. This adds available free nucleotides complementary to the DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure template strand, resulting in a newly synthesized DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure molecule.

Image by Lecturio.

Cycles repeat

  • Newly created DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure serves as the template for further PCR cycles.
  • Replicates in a 2X factor manner, where x is the number of replication cycles
  • The target DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure segment can be amplified 105–106-fold.

Analysis

  • The amplified DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure segment can then be observed with techniques such as gel electrophoresis.
  • Results should be compared with both:
    • Positive control
    • Negative control

Variations

There are many variations of the basic PCR technique. Some of the more common include:

  • Reverse transcription Transcription Transcription of genetic information is the first step in gene expression. Transcription is the process by which DNA is used as a template to make mRNA. This process is divided into 3 stages: initiation, elongation, and termination. Stages of Transcription PCR (RT-PCR)
  • Quantitative PCR (qPCR)

RT-PCR

  • Uses RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure as the template
  • Reverse transcriptase is used → create a complimentary DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure strand ( DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure/ RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure hybrid)
  • The RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure portion is degraded by reverse transcriptase or ribonuclease H.
  • DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure polymerase adds complementary nucleotides to the DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure strand → completed DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure molecule
  • The new DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure becomes the template → cycles through the normal PCR process from there

qPCR

  • Also called real-time PCR
  • Monitors amplification by attaching either: 
    • Fluorescent probe (sequence specific)
    • Fluorescent dye (intercalates with DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure)
  • Allows detection and visualization with specialized software
  • Useful for:
    • Quantification (e.g., viral load)
    • Quick detection of a targeted sequence (without requiring post-PCR analysis techniques)

Uses

Forensics

  • PCR allows comparison of small samples of DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure evidence from a crime scene with the DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure of suspects.
  • Paternity testing

Genotyping and sequencing

  • Identification of genetic mutations:
    • Allows for the diagnosis of many genetic diseases
    • Can be performed on embryonic cells to determine whether an embryo has a predisposition to certain conditions
    • Can identify genetic carriers → helpful for genetic counseling
  • Recognition of altered oncogenes can allow for targeted cancer therapy.
  • Can be used for tissue typing in organ implantation

Infectious disease

The PCR has significantly improved the diagnostic potential for infectious diseases.

  • Can rapidly determine the identity of microbes that were traditionally:
    • Slow growing on culture
    • Unable to be cultured
  • Examples:
    • HIV
    • HSV HSV Herpes simplex virus (HSV) is a double-stranded DNA virus belonging to the family Herpesviridae. Herpes simplex virus commonly causes recurrent infections involving the skin and mucosal surfaces, including the mouth, lips, eyes, and genitals. Herpes Simplex Virus 1 & 2
    • SARS-CoV-2
    • Influenza Influenza Influenza viruses are members of the Orthomyxoviridae family and the causative organisms of influenza, a highly contagious febrile respiratory disease. There are 3 primary influenza viruses (A, B, and C) and various subtypes, which are classified based on their virulent surface antigens, hemagglutinin (HA) and neuraminidase (NA). Influenza typically presents with a fever, myalgia, headache, and symptoms of an upper respiratory infection. Influenza Viruses/Influenza
    • Mycobacterium Mycobacterium Mycobacterium is a genus of the family Mycobacteriaceae in the phylum Actinobacteria. Mycobacteria comprise more than 150 species of facultative intracellular bacilli that are mostly obligate aerobes. Mycobacteria are responsible for multiple human infections including serious diseases, such as tuberculosis (M. tuberculosis), leprosy (M. leprae), and M. avium complex infections. Mycobacterium
    • Borrelia Borrelia Borrelia are gram-negative microaerophilic spirochetes. Owing to their small size, they are not easily seen on Gram stain but can be visualized using dark-field microscopy, Giemsa, or Wright stain. Spirochetes are motile and move in a characteristic spinning fashion due to axial filaments in the periplasmic space. Borrelia burgdorferi
  • qPCR allows for quantification of the microbial load (in addition to qualitative analysis).
  • Can also be used to detect genes encoding antibiotic resistance

Advantages and Disadvantages

Advantages

  • Rapid
  • Inexpensive
  • Relatively easy to use
  • Highly specific 
  • Sensitive (allows for use on very small sample sizes)
  • Can amplify DNA replication DNA replication The entire DNA of a cell is replicated during the S (synthesis) phase of the cell cycle. The principle of replication is based on complementary nucleotide base pairing: adenine forms hydrogen bonds with thymine (or uracil in RNA) and guanine forms hydrogen bonds with cytosine. DNA Replication exponentially to create an almost infinite number of copies of the target DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure
  • PCR machine replicates DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure in vitro:
    • No bacteria Bacteria Bacteria are prokaryotic single-celled microorganisms that are metabolically active and divide by binary fission. Some of these organisms play a significant role in the pathogenesis of diseases. Bacteriology: Overview required
    • No DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure extraction

Disadvantages

  • Extremely sensitive → prone to errors from RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure or DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure contamination
  • Primers require sequence data (can only be used to identify the presence of a known gene). 
  • Potential for primers to anneal to other similar sequences → amplification of the wrong gene sequence
  • Primer dimer formation:
    • Primers hybridize to each other via complementary bases.
    • Results in amplification of the dimer instead of the target sequence

References

  1. Fan, H, & Robetorye, RS. (2010). Real-time quantitative reverse transcriptase polymerase chain reaction. transcriptase-polymerase transcriptase-polymerase chain reaction. Methods Mol Biol. 630:199–213. http://reference.medscape.com/medline/abstract/20300999
  2. Brooks, GF CK, Butel, JS, Morse, SA, & Mietzneron, TA. Principles of diagnostic medical microbiology. In Brooks, GF CK, Butel, JS, Morse, SA, & Mietzneron, TA (Eds.), Jawetz, Melnick & Adelberg’s Medical Microbiology. New York: McGraw-Hill; 2010.
  3. Wittwer, CT, Herrmann, MG, Moss, AA, & Rasmussen, RP. (2013). Continuous fluorescence monitoring of rapid-cycle DNA amplification. Biotechniques. 54(6):314–20. https://pubmed.ncbi.nlm.nih.gov/23905170/
  4. Ishmael, FT, & Stellato C. (2008). Principles and applications of a polymerase chain reaction: Basic science for the practicing physician. Ann Allergy Asthma Immunol. 101(4):437–43. https://pubmed.ncbi.nlm.nih.gov/18939735/
  5. Smith, CJ, & Osborn, AM. (2009). Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 67(1):6-20. https://pubmed.ncbi.nlm.nih.gov/19120456/
  6. Ghannam, MG., & Varacallo, M. (2021). Biochemistry, polymerase chain reaction. [Online] StatPearls. Retrieved December 5, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK535453/
  7. Raby, BA. (2021). Tools for genetics and genomics: Polymerase chain reaction. In Tirnauer, JS (Ed.), UpToDate. Retrieved December 5, 2021, from https://www.uptodate.com/contents/tools-for-genetics-and-genomics-polymerase-chain-reaction
  8. National Human Genome Research Institute (2020). Polymerase chain reaction (PCR) fact sheet. National Institute of Health. Retrieved December 5, 2021, from https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet

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