Explain how a gene is amplified using PCR
Polymerase chain reaction (PCR) is a technique used to amplify a particular segment of DNA. PCR can be used to amplify a gene from genomic DNA or from a cDNA library. Here is the basic procedure for amplifying a gene using PCR:
1. Primer design: The first step in PCR is to design primers that will anneal specifically to the target gene. Two primers are designed that are complementary to sequences at the 5’ and 3’ ends of the target region. These primers will define the boundaries of the region to be amplified.
2. Denaturation: The genomic DNA or the cDNA is first heated to 96-98°C for 30 seconds to 2 minutes to denature the DNA into single strands.
3. Annealing: The temperature is then lowered to the annealing temperature (typically between 52-65°C) and the primers are annealed to the complementary sequences on the single-stranded DNA template.
4. Extension: The temperature is then raised to 72°C and Taq polymerase, a thermostable DNA polymerase, extends the primers along the template DNA in a 5’ to 3’ direction, adding nucleotides to the growing strand of DNA.
5. Amplification: The process of denaturation, annealing, and extension is repeated multiple times in cycles. Each cycle doubles the number of copies of the DNA segment, and after 20-40 cycles, the target gene will be amplified to a detectable level.
Overall, PCR is a powerful tool for amplifying a specific gene. It is widely used in genetic research, clinical diagnosis, forensic science, and many other applications.
PCR (Polymerase Chain Reaction) is a technique used to amplify a specific segment of DNA. Here's how a gene is amplified using PCR:
1. Denaturation: The DNA template containing the gene of interest is heated to high temperatures (typically around 94-98°C) to separate the DNA strands or denature the double-stranded DNA into single strands.
2. Annealing: The temperature is lowered to allow short DNA primers, which are specifically designed to bind to the DNA strands on either side of the target gene, to anneal or bind to their complementary sequences.
3. Extension or elongation: DNA polymerase enzyme is added to extend the bound primers along the DNA template. This step occurs at a temperature optimum for the DNA polymerase used, typically around 72°C. The polymerase copies the DNA starting from the primer and synthesizes a complementary DNA strand in the 5' to 3' direction.
4. Repetition: The Denaturation, Annealing, and Extension steps are repeated multiple times (usually 20-40 cycles) in a PCR machine called a thermocycler. Each cycle doubles the amount of DNA, resulting in an exponential amplification of the target gene.
By the end of the amplification process, billions of copies of the target gene are produced from the initial few molecules of DNA template. These amplified copies of DNA can then be used for various downstream applications in research, diagnostics, and genetic engineering.