Polymerase Chain Reaction (PCR): Difference between revisions

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=Definition=
=Description=
Polymerase chain reaction (PCR) using a method used to amplify a small amount of DNA in order to allow scientist to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [4]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are taken advantage of to recreate cDNA [4].
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 and converted into complimentary DNA (cDNA) with the use of a reverse transcriptase [4]. 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.  
 
==Primers==
==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. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,3,5,6]
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]
==Method==
There are three essential steps when conducting PCR.


1. The melting of the target DNA [2]
==Methods==
There are several essential steps when conducting PCR.  


2. After the DNA has been melted the primers are combined into a synthesized DNA [2]
1. Purifying RNA from specific tissues or cells [2]
 
2. Amplifying cDNA copies of the purified RNA [2]
 
3. Analysis of copied DNA sequences [2]


3. Finally, there is a primer extension by thermostable DNA polymerase [2]
[[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]]]
[[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]]]
==Stages of PCR==
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].
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].
These phases are repeated roughly 20-40 times, producing potentially billions of copies of the targeted sequence in a short period of time.


==Uses==
==Uses==
PCR is helpful for scientist 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 scientists to get a closer look at DNA [5]. Pathogens among samples are able to be seen using PCR [1,4,6]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].
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==
==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
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
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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.
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.
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.

Revision as of 20:12, 3 May 2021

Description

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 and converted into complimentary DNA (cDNA) with the use of a reverse transcriptase [4]. 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.

Primers

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

There are several essential steps when conducting PCR.

1. Purifying RNA from specific tissues or cells [2]

2. Amplifying cDNA copies of the purified RNA [2]

3. Analysis of copied DNA sequences [2]

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

Stages of PCR

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].

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].

These phases are repeated roughly 20-40 times, producing potentially billions of copies of the targeted sequence in a short period of time.

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.

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

4. 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

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.

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.

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