20: PCR
Summary
The polymerase chain reaction (PCR) is a method that amplifies a target DNA sequence.
Also known as
Polymerase chain reaction
Samples needed
Sample of DNA, which can be a mixture of sequences
Method
PCR is used to amplify, or make many additional copies of, a specific DNA sequence. PCR is used as a step in a wide variety of applications.
In order to perform a PCR, the following components must be included in the reaction mixture:
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A DNA sample containing the template DNA, i.e. the DNA sequence to be amplified
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A DNA polymerase enzyme
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A mixture of the four dNTPs
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Primers
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An appropriate buffer solution
The sequence amplified is determined by the use of primers: short oligonucleotides that anneal to specific DNA sequences. The DNA polymerase enzyme requires a primer to begin copying a DNA template, so it will only act on the 3’ end of the primer or primers provided by the researcher.
The reaction mixture is then subjected to a series of different temperatures, usually in a machine called a thermal cycler. Each cycle of PCR should double the amount of target DNA sequence present. Each cycle consists of three steps:
Denature template (separate DNA strands) at high temperature: mid 90s °C
Anneal primers at low temperature: Exact temperature depends on primers, but usually in 50s °C
Extend by DNA polymerase at moderate temperature: often 72 °C
Controls
Sometimes primers will anneal to a DNA sequence that is not an exact match, and therefore, the amplified DNA will not be the desired sequence. A DNA sample without the desired template DNA can be used as a negative control to confirm primer specificity.
Furthermore, sometimes a no template control (water instead of DNA sample) is used to ensure that none of the other reagents are contaminated with template DNA
Interpretation
In this experiment, researchers tackled the problem of genotyping transgenic mice with a mutant Atm (C3001L) allele integrated into a random genomic locus, i.e. an Atm transgene. To fully determine the Atm status of these mice, researchers need to know if each individual mouse has 0, 1, or 2 copies of the transgene (“transgene zygosity”), as well as whether the endogenous Atm locus contains 0, 1, or 2 copies of an Atm null allele. When referring to the endogenous Atm locus, “+” denotes wild type and “-” denotes the null allele. When referring to the transgene (Tg), “+/+” denotes two transgenes, “+/-” denotes one, and “-/-” denotes none.
Panel A in this figure shows the reader where the two primers bind, and therefore, which segment of DNA will be amplified during PCR: the section between primers 5 and 6. The three possible alleles are shown: the Atm transgene with a C3001L mutation, wild type Atm at the endogenous locus, and an Atm null allele, also at the endogenous locus. The amplicons of WT and Tg are the same size, so a further step must be taken to distinguish them by gel electrophoresis. Primer 6 does not bind to the null allele, so no amplification will occur from that template.
Anticipating the problem of distinguishing the WT endogenous Atm from the transgene, the researchers introduced an EcoRI restriction enzyme site into an intron in the transgene amplicon. To genotype mice, two steps are required. First, an experiment not shown here is used to determine the Atm genotype at the endogenous locus. Second, to determine how many copies of the transgene are present, PCR is performed with primers 5 and 6, followed by an EcoRI digest and gel electrophoresis . The number of transgene copies present is determined by comparing the intensity of the transgene bands to the WT band.
The results of such a genotyping experiment are shown in panel B. Remember that the genotype at the endogenous Atm locus is already known before the experiment shown. If the C3001L allele is present, two restriction fragments of 354 and 216 bp are expected due to EcoRI digestion. If the wild type allele is present, an intact amplicon of 570 bp is expected, as it lacks an EcoRI restriction site.
The number of transgenes can be determined by comparing the intensity of the band at 570 bp (WT) to the intensity of the bands at 354 & 215 bp (Tg). Consider control mouse C7. Researchers knew that it was +/+ at the endogenous locus and +/- at the transgene locus, and the Tg/WT ratio (for band intensity) was 1.32. All mice in this experiment except C5 are +/+ at the endogenous locus. Therefore, any unknown mouse with a Tg/WT ratio of ~1.32 is +/- at the transgene locus, as seen for instance in m14. Mice that are +/+ at the transgene locus will have a ratio roughly double that, as seen in m12 and m15. Any mice that are -/- at the transgene locus will lack the smaller bands entirely, as we see in m13. (To determine transgene zygosity in mice -/- at the endogenous locus the researchers used a different reverse primer that could bind the null allele.)
Image Descriptions
Figure 1 image description:
Panel A: A schematic of the genomic DNA of two Atm alleles: Atm TgC3001L and Atm WT. Both alleles show primer 5 (upstream) and primer 6 (downstream) pointing toward one another. Primer 5 binds in an intron, and primer 6 binds in exon 37. The amplicon contains an EcoRI restriction site only in the mutant allele.
Panel B: An image of DNA gel.
|
Mouse |
Endogenous Atm genotype |
570 bp band present? (Indicates WT) |
354 & 216 bp bands present? (Indicates Tg) |
Tg/WT ratio |
Sample type (Referring to Atm Tg) |
Atm Tg genotype |
|---|---|---|---|---|---|---|
|
C5 |
-/- |
N |
Y |
N/A |
Known-genotype control |
+/- |
|
C6 |
+/+ |
Y |
N |
0.00 |
Known-genotype control |
-/- |
|
C7 |
+/+ |
Y |
Y |
1.32 |
Known-genotype control |
+/- |
|
m12 |
+/+ |
Y |
Y |
2.30 |
Unknown transgene zygosity |
+/+ |
|
m13 |
+/+ |
Y |
N |
0.01 |
Unknown transgene zygosity |
-/- |
|
m14 |
+/+ |
Y |
Y |
1.27 |
Unknown transgene zygosity |
+/- |
|
m15 |
+/+ |
Y |
Y |
2.40 |
Unknown transgene zygosity |
+/+ |
Thumbnail
"PCR tubes.png" ↗ by Madprime is licensed under CC BY 1.0 ↗.
Description: Photo of a strip of PCR tubes, each tube contains a 1000uL (1mL) reaction.
Author
Katherine Mattaini, Tufts University
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Yang, J., A. N. DeVore, D. A. Fu, M. M. Spicer, M. Guo, S. G. Thompson, K. E. Ahlers-Dannen, F. Polato, A. Nussenzweig, and R. A. Fisher. 2023. Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method. Life Science Alliance 6: e202201729. ↵