Some of the major steps involved in polymerase chain reaction in DNA sequence are: 1.
Step 1: Denaturation by Heat 2. Step 2: Annealing Primer to Target Sequence 3. Step 3: Extension 4. Step 4: End of the First PGR Cycle.
The polymerase chain reaction (PGR) amplifies a single piece of DNA across several orders of magnitude, see figure 6.2.
It is the creation of thousands to millions of copies of a particular DNA sequence.
PGR is a three-step process or cycle. It is repeated a specified number of times.
Here are 8 important steps in the IPO process:
Once PGR cycle is comprised of these steps namely Denaturation, Annealing and Extension. An instrument automatically controls the process which takes place in a thermal cycler the temperatures alternates for programmed periods of time for the appropriate number of PGR cycles.
Step 1: Denaturation by Heat:
Heat is normally more than 90 degrees Celsius at separates double-stranded DNA into two single strands. This process is known as “denaturation”.
The hydrogen bonds linking the bases to one another are weak, therefore denaturation is possible. The hydrogen bonds break at high temperatures, whereas the bonds between deoxyribose and phosphates, remain intact as these are stronger remain intact.
Step 2: Annealing Primer to Target Sequence:
The ultimate goal of PGR is to replicate a target sequence of approximately 100-600 base pairs unique to the organism being studied.
Targeting the sequence is achieved by using primers. Primers mark the ends of the target sequence.
AMPLICOR® Test primers are short, synthetic sequences of single-stranded DNA typically consisting of 20-30 bases, with a biotin-labeled 5′ end to aid in detection.
For the target region of the organism, they are specific PGR has two primers, one for each of the complementary single DNA strands that was produced during denaturation single DNA strands that was produced during denaturation.
The beginning of the DNA target sequence of interest is marked by the primers that anneal (bind) to the complementary sequence.
Annealing usually takes place between 40 degrees Celsius and 65 degrees Celsius, depending on the length and base sequence of the primers.
Step 3: Extension:
The temperature is raised to approximately 72 degrees Celsius after the primers anneal to the complementary DNA sequences. Also, the enzyme Taq DNA polymerase is used to replicate the DNA strands.
Taq DNA polymerase is a recombinant thermo stable DNA polymerase from the organism Thermus aquatics us and unlike normal polymerase enzymes is active at high temperatures.
The synthesis process synthesizes new double stranded DNA molecules, both identical to the original double stranded target DNA region, by facilitating the binding and joining of the complementary nucleotides that are free in solution (dNTPs).
Extension always begins at the 3′ end of the primer making a double strand out of each of the two single strands. Taq DNA polymerase synthesizes exclusively in the 5′ to 3′ direction.
Step 4: End of the First PGR Cycle:
There are now two new DNA strands identical to the original target at the end of the first PGR cycle. The newly formed strands have a beginning, which is precisely defined by the 5′ end of the primer, but the 3′ end is not precisely defined.
The DNA strand synthesized from such a template then has a precisely defined length that is limited at either end by the 5′ end of each of the two primers.
Step 2: Getting the Prospectus Ready
These DNA strands are known as AMPLIGON. DNA strands which correspond to the target sequence. After only a few cycles, are present in much larger numbers then the variable length sequences.
The sequence flanked or defined by the two primers is the section that is amplified.