QUESTION: Which of the following is not an application of PCR?
A Molecular diagnosis
B Gene amplification
C Detection of gene mutation
D Purification of isolated protein
ANSWER:
Purification of isolated protein
| PCR |
Description |
| Full form of PCR |
· PCR stands for Polymerase Chain Reaction |
| Introduction |
· PCR is a laboratory technique used to amplify and create multiple copies of DNA from a single DNA segment. |
| Invention Year |
· PCR was invented in 1983 by Kary B. Mullis who was an American biochemist,
· He was awarded by a Nobel Prize in Chemistry in 1993 for this contribution. |
| Procedure |
· Denaturation: The DNA sample is first heated at a high temperature usually around 94-98°C to separate the double-stranded DNA into single strands.
· Annealing: During the process of annealing, temperature is lowered to 50-65°C to allow primers to bind to complementary sequences on the DNA strands.
· Extension: DNA polymerase adds nucleotides to the primers, synthesizing new DNA strands. This step occurs at a slightly higher temperature (usually 72°C). These steps are repeated in a cycle, leading to an exponential increase in DNA copies. |
| Components
|
· DNA template
· Primers
· DNA polymerase
· Nucleotides
· Buffer solution |
| PCR Machine |
· Thermal cycler or PCR machine is the Instrument that automates temperature changes during the PCR cycle.
· It controls the heating, cooling, and cycling required for denaturation, annealing and extension steps. |
| Applications of PCR |
· PCR is a powerful tool for various applications in molecular biology and genetics.
· Molecular Diagnosis: It is a key technique in clinical diagnostics, allowing early and accurate disease detection.
· Gene Amplification: PCR enables the rapid and efficient generation of multiple copies of specific genes, essential for gene analysis, sequencing, and cloning.
· Detection of Gene Mutation: PCR can detect genetic mutations associated with diseases and conditions, aiding in genetic screening and personalized medicine. |
| Types of PCR |
· Conventional PCR: Basic PCR with a fixed set of primers.
· Real-time PCR: Quantifies DNA as it amplifies, allowing for real-time monitoring of the process.
· Reverse Transcription PCR (RT-PCR): Converts RNA into complementary DNA before amplification.
· Multiplex PCR: Amplifies multiple targets in a single reaction.
· Nested PCR: Uses two sets of primers to increase specificity.
· Digital PCR: Divides the PCR reaction into many smaller reactions to quantify target DNA more accurately.
· Long PCR: Amplifies long DNA fragments. |
| Advantages |
· Rapid and efficient amplification of DNA.
· Requires small amounts of DNA for analysis.
· Versatile and widely applicable in various fields.
· High sensitivity and specificity when designed carefully. |
| Limitations |
· Susceptible to contamination due to its sensitivity.
· Requires careful primer design to avoid nonspecific amplification.
· Limited by the length of DNA that can be amplified in a single reaction.
· Some PCR inhibitors can interfere with the reaction.
· Artifacts and mutations can occur during the process. |
| Impact |
· PCR revolutionized molecular biology and genetics research.
· It enabled advances in genomics, forensics, medical diagnostics, evolutionary studies, and more. |
