Methods (molecular, IHC, frozen)
Molecular
PCR history & applications


Topic Completed: 1 January 2010

Minor changes: 22 March 2021

Copyright: 2008-2021, PathologyOutlines.com, Inc.

PubMed Search: Polymerase chain reaction[TI] history free full text[sb], Polymerase chain reaction[TI] current applications

Note: as PCR applications are voluminous, references are meant to be illustrative and not complete

Rodney E. Shackelford, D.O., Ph.D.
Page views in 2020: 143
Page views in 2021 to date: 77
Table of Contents
History | Current applications
Cite this page: Shackelford R. PCR history & applications. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/MolecularPCRhistory.html. Accessed April 15th, 2021.
History

Pre-PCR history
  • Common technique used to amplify very small amounts of a specific DNA sequence, even a single copy, into millions or up to 100 billion copies in a short time (Wikipedia: Polymerase Chain Reaction [Accessed 4 June 2018])
  • Technique works best on short DNA sequences of 100 to 1,000 base pairs and employs dNTPs, Taq DNA polymerase, oligonucleotides, specific buffer conditions and thermal cycling
  • PCR has many variations and is a fundamental tool in medical research and molecular pathology
  • Developed in 1983 by Kary Mullis, who won the Nobel Prize in Chemistry in 1993 for this work (Wikipedia: Kary Mullis [Accessed 4 June 2018])
  • Presently over 350,000 papers have been published which use or refer to this technique
  • Prior to PCR technology, obtaining multiple copies of a specific DNA sequence was commonly done but often laborious

  • Most protocols involved:
    1. Isolating many copies of the sequence(s) desired
    2. Cloning the DNA into a viral or bacterial plasmid vector
    3. Transfecting, selecting and growing the bacteria carrying the DNA and vector
    4. Reisolating the desired sequence(s) from bacterial cultures by purifying and cutting the plasmids

  • Limitations:
    1. Procedure could take several weeks
    2. Often difficult to get pure DNA / gene sequences from the complex mixtures typically used to obtain DNA samples

Initial discovery of PCR
  • Conceived in 1983 by Kary Mullis, working at Cetus Corporation as a chemist (Wikipedia: Kary Mullis [Accessed 4 June 2018])
  • Initial idea was to use a pair of primers to bracket the desired DNA sequence and to copy it repeatedly using DNA polymerase
  • Mullis received a $10,000 bonus from Cetus for the invention; Cetus later sold the patent to Roche for $300 million; Mullis may have received additional money for testifying on behalf of Cetus in a patent lawsuit

Later PCR related work
  • Cetus initially used PCR to detect the hemoglobin sickle cell point mutation
  • Mullis thought of using DNA polymerase from Thermophilus aquaticus (Taq), which was heat resistant; this eliminated the need to add enzyme after every cycle of thermal denaturation of the DNA, making the technique more affordable and subject to automation (Wikipedia: Thermus aquaticus [Accessed 4 June 2018])
  • Mullis won the Nobel Prize in Chemistry in 1993 for this work
Current applications

Archeology and evolution
  • DNA that has survived in ancient tissue up to 45,000 years old can be amplified to provide large quantities for sequencing
  • Analysis of DNA from ancient organisms is used to study ancient species, as well as evolution (Genome Res 1991;1:107)

DNA sequencing
  • Determining the order of DNA bases
  • Traditional Sanger method is not based on PCR but some newer sequencing methods use PCR to make copies of the DNA before the sequencing begins (Wikipedia: DNA Sequencing [Accessed 4 June 2018])
  • Although PCR introduces replication errors, DNA sequencing of the total PCR product may give the correct sequence because
    1. Errors occur in only a small percentage of the bases
    2. Incorporation of incorrect bases is essentially random
    3. New DNA polymerases have lower frequencies of mutations due to proofreading capabilities (Strachan: Human Molecular Genetics, 4th Edition, 2010)

Forensic identification
  • To identify individuals, forensic scientists scan 13 DNA regions or loci that vary from person to person and use the data to create a DNA profile of that individual ("DNA fingerprint")
  • Information is stored in the CODIS database, funded by U.S. FBI (Wikipedia: Combined DNA Index System [Accessed 4 June 2018])
  • There is an extremely small chance that another person has the same DNA profile for a particular set of 13 regions
  • PCR is used to make millions of exact copies of DNA from a biological sample, which allows use of biological samples as small as a few skin cells
  • Note: great care must be taken to prevent contamination with other biological materials during the identifying, collecting and preserving of a sample

Medical and pathogen diagnosis

Molecular genetics

Molecular pathology

Paternity testing
  • Use of genetic fingerprinting to determine if a man is the biological father of an individual
  • Current techniques use PCR and restriction fragment length polymorphisms
  • Older techniques used ABO blood group typing, analysis of other proteins or HLA antigens
  • In a DNA parentage test, the probability of parentage is 0% if the alleged parent is not biologically related to the child and typically > 99.9% if the alleged parent is biologically related to the child (Wikipedia: DNA Paternity Testing [Accessed 4 June 2018])

Tissue identification

Transplant engraftment analysis
  • Transplant engraftment studies are used to evaluate the level of donor versus recipient cells in posttransplant specimens
  • Unique DNA fingerprints from recipient and donor are used to determine the proportion of each contained within the total DNA extracted from the posttransplant specimen; these percentages correspond to relative amounts of donor and recipient cells in the specimen
  • Engraftment studies are sequentially performed on transplant patients to monitor closely the levels of donor and recipient cells so that appropriate therapeutic intervention can proceed, if necessary
  • Analysis often uses short tandem repeats (STRs) as part of the "fingerprint" (Bone Marrow Transplant 2002;29:243)

Tumorigenesis
Back to top
Image 01 Image 02