Thermal Cycler
PCR is a molecular biology technique utilized to amplify small sections of DNA or a gene, enabling the generation of numerous copies of a specific DNA segment from a minuscule initial quantity. The polymerase chain reaction (PCR) was initially created in 1983 by Kary Mullis, an American biochemist who was subsequently awarded the Nobel Prize in Chemistry in 1993 for his groundbreaking contributions. During the COVID-19 pandemic, PCR was employed as a crucial part of testing procedures to confirm the presence of the virus in infected individuals.
PCR, which stands for polymerase chain reaction, is a method employed in medical and molecular biology research to produce a large number of copies, ranging from thousands to millions, of a specific section of DNA, such as a particular gene. Its applications are diverse, including the initial steps of DNA processing for sequencing purposes and the creation of forensic DNA profiles using minute quantities of DNA. Additionally, it can be utilized for gene detection to aid in the identification of pathogens during infections. For instance, during the COVID-19 pandemic, PCR tests were utilized to identify the presence or absence of a segment of the virus's genetic material in a person's swab sample, providing a positive or negative result for infection.
1.The DNA template that serves as the target for replication.
2.Primers, which are short segments of DNA or RNA, typically 20 to 30 bases in length. These primers bind to both ends of the DNA region of interest, indicating the starting point for PCR.
3.DNA nucleotide bases, also known as dNTPs, which are the building blocks of DNA. These bases (A, T, C, and G) are necessary for constructing the new DNA strand during PCR.
4.Taq polymerase, an enzyme responsible for adding the nucleotide bases to the replicated DNA sequence.
5.A buffer solution that creates the optimal conditions for the PCR reaction to occur.
The double-stranded template DNA is subjected to high temperatures, causing it to separate into two single strands.
The temperature is lowered, facilitating the attachment of DNA primers to the template DNA.
The temperature is raised again, and the Taq polymerase enzyme synthesizes a new DNA strand.
These three stages are repeated 20-40 times, resulting in the doubling of the DNA copies with each cycle. This process, known as thermal cycling, is performed by a machine called a thermal cycler. Depending on the machine's speed, PCR can be completed in less than an hour or take a few hours.
Following the completion of PCR, a technique called electrophoresis can be employed to assess the quantity and size of the produced DNA fragments.
•During this stage, the reaction mixture is subjected to a temperature of 94-95⁰C for approximately 15 to 30 seconds.
• The elevated temperature leads to the disruption of hydrogen bonds between the bases in the two strands of template DNA, causing the strands to separate.
• As a result, two individual DNA strands are formed, serving as templates for the synthesis of new DNA copies.
• It is crucial to maintain the temperature at this stage for an adequate duration to ensure complete separation of the DNA strands.
• In this stage, the reaction is cooled down to facilitate the attachment of primers to a specific location on the single-stranded template DNA through hydrogen bonding.
• The temperature required for annealing depends on the primer's characteristics, typically ranging between 50 and 65⁰C.
• The two separated DNA strands are complementary and oriented in opposite directions, with one end being the 5' end and the other being the 3' end. Consequently, two primers are used: a forward primer and a reverse primer.
• This step holds significant importance as the primers establish the starting point for DNA synthesis. By providing a short region of double-stranded DNA, they enable the polymerase enzyme to initiate the creation of the new complementary DNA strand using the available loose DNA bases in the subsequent extension step.
• Typically, the annealing step lasts approximately 10-30 seconds.
• The temperature is raised to 72⁰C to facilitate the synthesis of new DNA strands using a specialized enzyme called Taq DNA polymerase, which adds DNA bases.
• Taq DNA polymerase is derived from the bacterium Thermus aquaticus, commonly known as "Taq." This bacterium naturally inhabits hot springs and exhibits tolerance to temperatures exceeding 80⁰C, with its optimal temperature being 72⁰C.
• The DNA polymerase extracted from this bacterium demonstrates exceptional stability at high temperatures, enabling it to withstand the temperature conditions required to separate the DNA strands during the denaturation stage of PCR.
• In contrast, DNA polymerase from most other organisms would not be able to endure such high temperatures. For instance, human polymerase functions optimally at 37˚C, which is equivalent to body temperature.
• At a temperature of 72⁰C, the Taq polymerase initiates the construction of the complementary DNA strand. It first binds to the primer and then sequentially adds DNA bases to the single strand in the 5' to 3' direction.
• This process leads to the formation of a completely new DNA strand and ultimately results in a double-stranded DNA molecule.
• The duration of this step varies depending on the length of the amplified DNA sequence. On average, it takes approximately one minute to replicate 1,000 DNA bases.
• The thermal cycling process is repeated 20-40 times, allowing for the generation of numerous copies of the desired DNA sequence.
• The newly synthesized DNA fragments produced during each cycle serve as templates for the attachment of the DNA polymerase enzyme, initiating further DNA synthesis.
• This repetitive process results in a substantial quantity of copies of the targeted DNA segment being generated within a relatively brief timeframe.
PCR finds applications in various fields of biology and medicine, including molecular biology research, medical diagnostics, and certain branches of ecology.
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