Polymerase Chain Reaction (PCR): Difference between revisions

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=Description=
=Background=  
The polymerase chain reaction (PCR) is a method used to amplify a small amount of DNA in order to allow scientists to study target genes or specific DNA sequences in detail [1]. DNA is extracted from sample tissues or cells and converted into complimentary DNA (cDNA) [4]. In some cases, RNA is extracted and converted to cDNA with the use of a reverse transcriptase. Target primers identify the location of the DNA sequence(s) in the sample, in which DNA polymerases recognize and begin to synthesize the complementary strands of targeted sequences. With each cycle of the reaction, cDNA begins to amplify exponentially at a point known as the cycle threshold value in which the abundance of cDNA can be measured using an instrument known as a thermocycler.
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for the creation of PCR along with Michael Smith for his work on PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].
 
==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]


==Primers==
==Primers==
[[File:EVS463_Phases_of_PCR.JPG|right|450px|thumb|The three phases of polymerase chain reactions [7]]]
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning and end, where the DNA-Polymerase binds and starts synthesis of new DNA strand [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].
PCR primers are single strands of RNA that recognize and attach to the targeted sequence of DNA in the sample tissue. Once the targeted sequence has been located, a DNA polymerase attaches to the primer and begins synthesizing cDNA strands, amplifying the target sequence. For bacteria and [[archaebacteria]], primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,3,5,6]


==Methods==
[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]
There are several essential steps when conducting Standard PCR.


1. Purifying DNA from specific tissues or cells [2]
==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100<sup>oC</sup> to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].


2. Amplifying cDNA copies of the purified DNA [2]
'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50<sup>oC</sup> below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]


3. Analysis of copied DNA sequences [2]
'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].


These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.


There are also a number of different types of polymerase chain reactions including endpoint, quantitative real time, reverse transcription, multiplex, and more. These have been modified to analyze different types of data, such as end point PCR where analysis of cDNA takes place after the plateau phase, or RT-PCR where cDNA is synthesized from RNA through the use of a reverse transcriptase enzyme.
[[File:PCR cyclePM.png|thumb|left|Multiple completed cycles of PCR [7]]]


==Stages of PCR==
==Uses==
[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|300px|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. It allows for the understanding of gene expression either spatially or temporally among [[organisms]] [1]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1].  Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1]. Bacterial cultures do not work well for slow-growing [[microorganisms]] such as mycobacteria and anaerobic bacteria [5]. PCR is able to be done rapidly and effectively making it a common practice across the science community. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Today PCR is widely known for detecting pathogens such as Covid-19.
1. '''Denaturing stage''': During this phase, the purified sample containing the double stranded (ds) DNA and reaction mixture is heated to a temperature of 94C-95C, breaking the hydrogen bonds and separating the strands to allow for future amplification [7].  


2. '''Annealing stage''': During this phase, the reaction mixture is cooled to a temperature of 50C-65C, allowing specific target primers (forward and reverse) to attach to the complementary target DNA sequence through hydrogen bonding. This step is necessary, as DNA polymerases cannot extend the primers to create new copies of the DNA without a section of dsDNA to begin with [7].
==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992


3. '''Extension stage''': During this phase, the temperature is increased to 72C to enable to attachment and activity of the DNA polymerase. This specific DNA polymerase comes from the heat-loving bacteria ''Thermus aquaticus'', which is stable at higher temperatures needed for the initial denaturing of double stranded DNA from sample tissues. Once this binds to the forward or reverse primer of the target DNA sequence, it begins to synthesize new strands or copies of the sequence through addition of dNTPs in the 5' to 3' direction [7].  
[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.


These phases are repeated roughly 20-40 times, producing potentially billions of copies of the targeted sequence in a short period of time.  
[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test
 
==Uses==
Polymerase chain reactions are helpful for scientists as they enhance specific target sequences within the genome of an organism. This can allow scientists to determine the temporal and/or spatial expression of genes throughout an organism, or even the differences between mutant and wild-type plants and [[animals]]. Today, PCR is commonly used in identifying pathogens among samples, such as COVID-19 and many other life-threatening viruses [1,4,6].
 
==References==
1. Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992


2. Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.
[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992


3. Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992
[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682


4. Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5
[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5


5. Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992
[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/


6. WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.
[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992


7. “What Is PCR (Polymerase Chain Reaction)?” Facts, The Public Engagement Team at the Wellcome Genome Campus, 25 Jan. 2016, www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction.
[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Latest revision as of 10:39, 7 May 2021

Background

Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for the creation of PCR along with Michael Smith for his work on PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].

Definition

PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

PCR Components

In order to conduct PCR there are numerous components that are required.

  • DNA template or cDNA: These are regions that will be getting amplified [5]
  • Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
  • Taq polymerase: This copies the region being amplified [5]
  • Nucleotides: This is from the DNA polymerase for the new DNA [5]
  • Buffer: This provides a chemical environment for the DNA-Polymerase [5]

Primers

PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning and end, where the DNA-Polymerase binds and starts synthesis of new DNA strand [1,2,4,8]. For bacteria and archaebacteria primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

Stages of PCR and the resultant amplification of DNA copies of the target region[2]

Stages of PCR

Denaturing Stage: The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

Annealing Stage: During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

Extension Stage: Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

Multiple completed cycles of PCR [7]

Uses

PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic ecology studies as it allows a closer look at DNA [8]. It allows for the understanding of gene expression either spatially or temporally among organisms [1]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in soil, sand, and sediment [1]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1]. Bacterial cultures do not work well for slow-growing microorganisms such as mycobacteria and anaerobic bacteria [5]. PCR is able to be done rapidly and effectively making it a common practice across the science community. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Today PCR is widely known for detecting pathogens such as Covid-19.

References

[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

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