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. |