PCR definition:
A common genetic tool- a laboratory technique used to obtain multiple
copies of target DNA fragments using Taq DNA polymerase in a
temperature-dependent reaction is called a PCR- a polymerase chain
reaction.”
Theoretically, the definition of the PCR can be as stated,
“PCR is a technique in which using the dNTPs, primers, Taq DNA
polymerase, and template DNA, artificial gene synthesis can be done.”
In short, we can define PCR as,
“An in vitro DNA amplification or synthesis technique is known as PCR.”
PCR requirements:
Chemicals:
dNTPs, distill water, PCR reaction buffer, enzyme Taq DNA polymerase, primers,
and template DNA.
Instruments:
Thermocycler, spinner and agarose gel electrophoresis unit.
Other utilities:
PCR tubes, stands, pipettes, tips.
PCR steps:
Various temperature zone governs each PCR step, viz denaturation,
annealing, and extension followed by a single initial denaturation and final
extension steps. In each step, different reactions occur.
PCR step 1: Denaturation
Temperature: 90°C to 95°C
Time 30 sec to 90 sec
In a denaturation two single-stranded DNA forms from the double-stranded
one. At 94ºC temperature, the double-stranded DNA opens up by breaking
hydrogen bonds. The process of denaturation is followed by the initial
denaturation for 5 to 7 minutes at the same temperature.
PCR step 2: annealing
Temperature: 55°C to 65°C
Time: 30 to 60 sec
After the denaturation, primer anneals to ssDNA at its exact annealing
temperature. Base on the GC content of primers, every primer has its own
annealing temperature.
The annealing temperature is usually ranging from 55ºC to 65ºC. Annealing
temperature lower than that leads to non-specific bindings while higher
temperature leads to amplification failure. 45 seconds to 1 minute are enough for
the second step, annealing for more than 1 minute causes non-specific
amplification.
PCR step 3: extension:
Temperature: 70°C to 72°C
TIme: 45 Sec
After the binding of the primer, its time to expand the DNA strand. Here in
extension step the Taq DNA polymerase comes in action and adds dNTPs to the
DNA strand. The temperature for the extension is 72ºC for 45 seconds.
After completing all steps one more time the final extension is performed
for 7 minutes.
Time-duration for PCR:
1 hour to 4 hours
PCR reagents:
Template DNA, PCR primers, dNTPs, Taq DNA polymerase and PCR buffer
are the major reagents of PCR reaction. The composition and quantity of
each reagent are very important. A single μL variation in any of the
reagents leads to reaction failure.
Template DNA:
The template must be DNA only. Plasmid DNA, bacterial DNA, cDNA, or
gDNA can be utilized as a template. The template DNA should be a highly
purified DNA that has a purity of around ~1.80 and a quantity of up to
200ng. The DNA works as a substrate for an enzyme when it denatured.
The good quality of extracted DNA can boost the resulting efficiency of the
polymerase chain reaction. The ideal concentration of gDNA for PCR
reaction is 30ng with 260/280 absorbance ration of ~1.80.
Concentration: 30 to 50ng.
PCR Primer:
Another important PCR ingredient is PCR primers. RNA primer governs the
replication reaction, normally, however, DNA primers are utilized during
PCR instead of RNA primers. The Taq DNA polymerase doesn’t have proof-
reading activity thus it can’t remove RNA primers. Probably, this is the
reason, we are using DNA primers in PCR.
The RNA primer is replaced during the proofreading activity in the
replication which is not possible in the case of Taq DNA Polymerase.
The PCR primers are artificially synthesized oligonucleotide sequences of
DNA ranging from 18 to 22 bases in length, short DNA sequences which
anneals at the single-stranded template DNA at its exact complementary
position.
For increasing the efficiency of primers, we should follow proper guidelines
while creating primers. GC content, melting temperature, length, and
primer-complementation capacity of primers are key factors for primer
[Link], 10pmol of each primer is sufficient for a PCR reaction.
Concentration: 10 to 12pMol.
dNTPs:
Deoxynucleotide triphosphates are artificially synthesized nucleotides
which bind to the growing DNA strand. With the help of the Taq DNA
polymerase, the dATP, dGTP, dCTP and dTTP binds at its complementary
nucleotides on the growing DNA strand.
1mM to 2mM of each dNTPs are sufficient for 25μL of PCR reaction.
Concentration: 200-250μM each.
Taq DNA polymerase:
The PCR technique is entirely based on the activity of Taq DNA polymerase.
If Taq DNA polymerase was not discovered, the PCR might not be
discovered.
Thermostability, the unique property of the Taq makes amplification
possible during PCR. Thermostability means it can work finely at a higher
temperature. Notably, no other bodily enzyme can function at a higher
temperature more than 37ºC.
The Taq DNA polymerase settles at the ssDNA- primer junction and utilizes
it as a substrate for the catalytic reaction. In the final step of extension,
using the substrate it starts dNTP insertion.
1 unit of Taq is sufficient for a 25μL PCR reaction.
Concentration: 1 to1.5 unit.
PCR buffer:
PCR enhancers help to boost reaction and amplification efficiency thus
PCR buffer is as important as other ingredients. Additionally, the PCR
buffer maintains the constant pH of the reaction nearly 7.9 to 8.5 by
keeping the constant chemical environment for the PCR reaction.
The pH of the buffer is controlled by the addition of Tris.
Mgcl2, DMSO, KCl, albumin, betaine, BSA, glycerol, (NH4)2SO4, and
formamide are some of the chemicals commonly used in the PCR buffer.
The composition of each ingredient may vary from manufacturer to
manufacturer.
However, in each PCR buffer, the MgCl2 must be included because it is
worked as a cofactor for the Taq DNA polymerase.
Concentration: 1X or as per requirement.
The PCR machine
The PCR machine is known as a thermocycler. This machine is simply a
heating block (just like our iron) which provides the constant temperature
and even rapidly changes between two temperature states.
The machine has a lower block of metal having deep wells for putting PCR
tubes. Also, the temperature of the inner environment is maintained by the
heating block present on the upper side of the lead.
Further, the machine contains the display, power on and off switch, and
cooling assembly. The machine has the ability to heat and cool the PCR
tube in a short period of time.
PCR procedure/ protocol:
Pre-preparation:
For any molecular genetic experiment, pre-preparation plays an important role in
getting good results.
Before starting the reaction, one must have to be ready for doing the lab
work, for that, wear a lab coat, gloves, a mouth cap, and a head cap.
Clean the PCR reaction preparation area and arrange all other utilities
nearby the reaction preparation.
Now take reagents from the deep freeze and thaw all the reagents properly.
Reaction preparation:
Take a sterile PCR tube and start adding reagents as shown in the table.
starts adding reagents in a sequential manner to reduce the chance of error.
If you have a ready to use mastermix, you can add it directly, this will save time
and increases the efficiency of the reaction.
After the completion of reaction preparation, close all the tube caps and spin it
properly, so that all the reagents mix well.
Now put the tubes in the PCR machine one by one in the pre-set PCR protocol.
Remember: don’t waste time setting protocol during the PCR, set it before the
reaction preparation, and immediately run the PCR.
Meanwhile start preparing the gel for agarose gel electrophoresis, because it will
also take time for around 60 to 90 minutes.
Post-preparation:
After completion of the PCR reaction, turn off the machine and collect all
the tubes in an “orderly manner”.
Rest tubes for some time in a freeze before doing agarose gel
electrophoresis.
You can also rest it for the next day, no problem with it.
Application of PCR
Diagnosis of inherited disease:
The PCR is most routinely used in the diagnosis of some inherited disease
such as sickle cell anemia, thalassemia, MTHFR gene mutation, etc. This
technique is appropriate for single-gene disorders. The result is 99%
accurate as compared with other methods.
Microbial identification:
The microbial culture technique is traditional and time-consuming and the
chance of infection is also high in the case of culturing. In modern days,
PCR is used in the identification of microbes. The bacteria’s unique DNA
sequence is targeted for the identification of particular bacteria. It will give
a result within 3 to 4 hours.
Additionally, PCR is also applicable to the diagnosis of infectious diseases
such as HIV or HPV. Again the method is the same as the identification of
microbes. The unique DNA sequence of a particular virus is targeted for
the identification. This will give a result within an hour.
PCR is used in the identification of genetic carriers as well. The
heterozygous condition of the disease can be easily identified using PCR
amplification.
DNA fingerprinting and genetic imprinting:
The PCR is the first choice for DNA fingerprinting Criminal verification,
identification of a person, and material cell contamination can be detected
using DNA fingerprinting.
The PCR is one of the best techniques for marker assistant selection.
RFLP, AFP, RAPD, STS, VNTR, and STR are some of the PCR based
techniques.
PCR is applicable in the prenatal diagnosis of inherited disease as well.
PCR helps in detecting cancer genes and infections.
Further PCR is applicable to sex determination and sex identification.
Apart from mutation detection, PCR is useful in gene expression studies
too. The expression of a particular gene can be measured using RT PCR. It
is even applicable in gene cloning.
mRNA studies are also possible due to the reverse transcriptase PCR and
we can calculate gene expression through it.
PCR amplification is one of the important steps in DNA sequencing and
microarray.
The PCR is also useful in the validation of personalized medicines.
The PCR is used in;
Gene editing
Gene manipulation
Genetic engineering
RNAi research
DNA and RNA quantification
cDNA and gDNA library preparation
Developing new assays
Limitations of PCR
Novel mutations can not be found using PCR, we have to do sequencing for
that.
Also, Multigenic disorders cannot be detected using PCR.
We can not identify structural and numerical chromosomal anomalies
through PCR.
Types of PCR:
1. Real-time PCR
2. Quantitative real time PCR (Q-RT PCR)
3. Reverse Transcriptase PCR (RT-PCR)
4. Multiplex PCR
5. Nested PCR
6. Long-range PCR
7. Single-cell PCR
8. Fast-cycling PCR
9. Methylation-specific PCR (MSP)
10. Hot start PCR
11. High-fidelity PCR
12. In situ PCR
13. Variable Number of Tandem Repeats (VNTR) PCR
14. Asymmetric PCR
15. Repetitive sequence-based PCR
16. Overlap extension PCR
17. Assemble PCR
18. Intersequence-specific PCR(ISSR)
19. Ligation-mediated PCR
20. Methylation –specifin PCR
21. Miniprimer PCR
22. Solid phase PCR
23. Touch down PCR, etc