How is a PCR Done: Polymerase Chain Reaction is a biochemical technique that is used in molecular biology. It is a quick way to amplify a minute quantity of DNA to obtain millions of copies of DNA molecules. Importantly, it allows researchers/scientists to take a small piece of DNA and amplify it to a large amount to study in detail. It was developed by Karry Mullis in 1983. He got a Nobel Prize in 1993. The first PCR machine was introduced in 1988. In this article, we will cover the most important aspects.
How is a PCR Done & Its Components
The following are four key components that need to be used in this process.
- Primer
- DNA Sequence
- Nucleotides
- The Enzyme (Thermus Aquaticus)
Primer: It is a short single strand of DNA sequence used to initiate DNA synthesis.
DNA Sequence: It is the process of determining the order of DNA sequence. Typically, it is used to identify nucleic acid sequences such as adenine, guanine, cytosine, and thiamine. Hence, a specific DNA segment is amplified enzymatically through Taq DNA Polymerase using Polymerase Chain Reaction.
Nucleotides: It is consists of adenine, guanine, cytosine, and thiamine. These are added to the PCR reaction to construct a new strand of DNA.
Taq Polymerase: It is the most common enzyme used for PCR amplification. The enzyme is heat resistant with a half-life of 40 minutes of 95°C. Polymerase chain reaction amplification works on the principle of temperature variation (heating and cooling reactions). Therefore, using this enzyme is very beneficial as it can work with high efficiency at high temperatures.
How is a PCR Done
Temperature plays an important role in the Polymerase chain reaction. Therefore, it uses three temperatures used for different purposes: denaturation, annealing, and expansion.
- Denaturation
High temperature (91-94°C) is used to denature double strands of the template DNA and separate them into single strands.
- Annealing
Low temperature (55 ° C) is used to attach the primer (short single-stranded DNA segment) to the template DNA.
- Extension
DNA Taq Polymerase (an enzyme) is used to attach the new strand of DNA at 71-72°C of temperature.
Read more: How does the test work?
Types of Polymerase Chain Reaction
How is a PCR Done: There are different types of PCR which are as follows.
- Inverse
This type of PCR is commonly used to amplify unknown DNA segments. It is also used to amplify transposable elements and in the process of mutagenesis. However, we need a known DNA sequence to identify unknown DNA segments using the restriction endonuclease enzyme to generate overhang. It will allow DNA to pair up with the alternate sequence. Now, the restriction enzyme will be reused to make the blunt ends circular. Then, it will lead to PCR sequencing.
- Colony
How is a PCR done using a colony method? It is used for the screening of recombinants from bacterial, bacteriophage, or yeast transformation products. So, there are a few steps involved in this process which are as follows.
- Cells are grown on the agar plates or in the flasks
- Collection of the colony in PCR tubes
- Place them inside the PCR machine and run until they are boiled for about 5-10 mins and then cool down
- Add additional PCR material such as dNTPs, enzymes, etc.
- Transfer to the DNA gel analysis
- You are ready to visualize the desirable segment
- Nested
A question is being raised on a large scale how is a PCR done using a Nested method. Nested holds limited contamination. As we know, the primer in the general method binds in the wrong way that amplifies a sequence incorrectly.
Fortunately, Nested PCR uses double primers (2-sets) that improve the activity of the reaction. It increases the sensitivity by reamplifying from a template previously enriched by the first PCR.
- Multiplex
It is a modification of the normal Polymerase chain reaction. It can amplify multiple targets in a single reaction. Hence, we need primers in different phases regarding amplifying the sequences on equal annealing temperatures. Then, it needs to be transferred to DNA gel electrophoresis. It can also be used to amplify complete genome targets.
- How is a PCR done with a Real-Time Method?
It is an in vitro (laboratory) technique used to detect the amplification of the target DNA molecules during the PCR reaction. In this molecular biology technique, DNA detection and amplification are measured throughout the process before it goes ends.
- Hot Start
It is often used to suppress enzymatic activity, usually the Taq polymerase enzyme. Hot start PCR is a variant of the polymerase chain reaction. It is a combination of the Taq polymerase enzyme and an aptamer-based inhibitor. Therefore, the inhibitor binds reversibly to the enzyme and inhibits its activity at 45 ° C temperature. But, it doesn’t restrain the enzyme during normal cyclic conditions.
- Touch down
This method is used to increase the specificity and sensitivity of the reaction. It uses a cyclic program where the annealing temperature is gradually decreased (1-2 ° C) after every second cycle. However, it is a method to decrease off-target priming to improve reaction activity.
- How is a PCR done In Situ Method?
This is a new molecular PCR technique. It detects minute quantities of single and rare copy numbers of nucleic acid sequences. Alternatively, it detects frozen or paraffin-embedded calls/tissues for the localization within the cells.
- Long Range
This method is used for the amplification of DNA length that can not be achieved using general or routine PCR methods. However, simple targeted sequences can be amplified up to 30 kb and beyond. Whereas, 20 kb is ideal for the complex genomic template.
Conclusion
Finally, we reached the end and knew how is a PCR done by different methods. In addition, we realized how important each of them was in different aspects. Besides, the polymerase chain reaction is a widely used technique nowadays.
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