POLYMERASE CHAIN REACTION
(PCR)
PCR is an abbreviation for "Polymerase Chain Reaction." . This term applies to a wide variety of different DNA tests that differ in reliability and effectiveness. Reliabilities of each kind of PCR test need independent verification. PCR itself doesn't accomplish DNA typing, it only increases the amount of DNA available for typing.
PCR uses constant regions of DNA sequence to prime the copying of variable regions of DNA sequence. [28]
PCR uses constant regions of DNA sequence to prime the copying of variable regions of DNA sequence. [28]
PCR typically uses two short pieces of known DNA called Primers . These serve as starting points for the copying of a region of DNA. [23]
PCR- THERMOCYCLER
The ThermoCycler is a laboratory apparatus used to amplify segments of DNA via the polymerase chain reaction (PCR) process.The device has a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps. [24]
PROCESS...
The advantage of using PCR for DNA fingerprinting is that a very small amount of DNA sample is required for the production of DNA fingerprint.
It works on the principle of DNA replication. It amplifies a specified region of 150 - 3,000 base pairs in length. [25]
As shown in the diagram alongside, PCR is carried in three steps.
1. INITIATION STEP-
It is the first step of the cycle which consists of raising the temperature of the reaction to 94–96 °C or 98 °C if extremely thermostable polymerases are used, which is held for 1–9 minutes. This process activates the DNA polymerase used in the reaction.
2.DENATURATION STEP-
It consists of heating the reaction to 94-98 degree centigrate for 20-30 seconds. This helps in breaking of the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
3. ANNEALING STEP-
The mixture is now cooled to a temperature of 50–65 degree centigrate for 20-40 seconds which helps in annealing of the primers to the single-stranded DNA template. The duration of annealing step is usually 1 min during the first as well as the subsequent cycles of PCR. Since the primer concentration is kept very high relative to that of the template DNA, primer-template hybrid formation is greatly favored over re-annealing of the template strands.
4 EXTENSION STEP-
It is a DNA polymerase dependent process. The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80 degree centigrade. The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing the 3′-OH of the primer. The primers are extended towards each other so that the DNA segment lying between the two primers is copied; this is ensured by employing primers complementary to the 3′-ends of the segment to be amplified. The duration of primer extension is usually 2 min at 72°C. Taq polymerase usually amplifies DNA fragments of up to 2 Kb; special reaction conditions are necessary for the amplification of longer segments. As a thumb rule, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute, leading to exponential (geometric) amplification of the specific DNA fragment.
5. FINAL ELONGATION-
This step is performed at a temperature of 70-74 degree centigrade for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
6. FINAL HOLD-
In this step the mixture is allowed to cool to a temperature of 4-15 degree centigrade for short-term storage of the reaction. [25]
It works on the principle of DNA replication. It amplifies a specified region of 150 - 3,000 base pairs in length. [25]
As shown in the diagram alongside, PCR is carried in three steps.
- Denaturation
- Annealing
- Extension
1. INITIATION STEP-
It is the first step of the cycle which consists of raising the temperature of the reaction to 94–96 °C or 98 °C if extremely thermostable polymerases are used, which is held for 1–9 minutes. This process activates the DNA polymerase used in the reaction.
2.DENATURATION STEP-
It consists of heating the reaction to 94-98 degree centigrate for 20-30 seconds. This helps in breaking of the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
3. ANNEALING STEP-
The mixture is now cooled to a temperature of 50–65 degree centigrate for 20-40 seconds which helps in annealing of the primers to the single-stranded DNA template. The duration of annealing step is usually 1 min during the first as well as the subsequent cycles of PCR. Since the primer concentration is kept very high relative to that of the template DNA, primer-template hybrid formation is greatly favored over re-annealing of the template strands.
4 EXTENSION STEP-
It is a DNA polymerase dependent process. The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80 degree centigrade. The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing the 3′-OH of the primer. The primers are extended towards each other so that the DNA segment lying between the two primers is copied; this is ensured by employing primers complementary to the 3′-ends of the segment to be amplified. The duration of primer extension is usually 2 min at 72°C. Taq polymerase usually amplifies DNA fragments of up to 2 Kb; special reaction conditions are necessary for the amplification of longer segments. As a thumb rule, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute, leading to exponential (geometric) amplification of the specific DNA fragment.
5. FINAL ELONGATION-
This step is performed at a temperature of 70-74 degree centigrade for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
6. FINAL HOLD-
In this step the mixture is allowed to cool to a temperature of 4-15 degree centigrade for short-term storage of the reaction. [25]
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